The rise of antibiotic resistance in bacterial pathogens is reducing the effectiveness of essential antibiotics used in invasive surgery and chemotherapy, complicating treatment for normally manageable diseases. To address this, developing new antibacterials and identifying molecules that enhance the efficacy of existing antibiotics are critical priorities.

This study evaluated the antimicrobial responses of various bacterial strains, particularly focusing on the potential synergistic effects of DFNAC and CBD while considering drug repurposing [41] and previous research [42,43]. Some findings suggest that CBD acts as a “potentiator,” improving standard antibiotics’ effectiveness against resistant strains, while others indicate that it may be an effective standalone antibacterial agent for topical use [44]. Other studies highlight DFNAC’s notable antibacterial and anti-inflammatory effects [45]. Our results demonstrated the synergistic interaction against Staphylococcus epidermidis when combining DFNAC and CBD, significantly enhancing antibacterial efficacy and substantially reducing the minimum inhibitory concentration (MIC). Conversely, when tested against Staphylococcus aureus, the combination exhibited an additive effect, indicating partial cooperation between DFNAC and CBD.

Indeed, research has been directed at identifying antimicrobial candidates to combat the rising antibiotic resistance in Gram-positive bacteria. Given the serious infections caused by Staphylococcus aureus, investigating natural products like CBD [36,46,47,48] is a logical step, and further evidence of in vivo efficacy is warranted [49,50]. However, pharmacodynamic studies are required to assess drug–drug interactions for a continuous evaluation of CBD’s benefits [43,44]. For instance, a four-fold reduction in the minimum inhibitory concentration (MIC) shows that only a small amount of CBD is needed to inhibit bacterial growth, highlighting its efficacy, especially in combination therapies or as a structural derivative. This reduction is crucial for reviving susceptibility in antimicrobial-resistant strains, broadening CBD’s therapeutic potential even when used alone. Moreover, a lower active dose reduces cytotoxicity risks and enhances safety. CBD has proven safe for human keratinocytes and erythrocytes at concentrations well above its antibacterial MIC, providing a significant margin of safety. However, achieving effective CBD concentrations in vivo depends on the method of administration. Topical administration is especially effective against Staphylococcus epidermidis, often related to skin and surgical infections, as creams can deliver CBD directly to the site, achieving necessary MICs. In contrast, systemic administration for internal infections can be challenging. Oral routes struggle with low bioavailability (about 10–20%) due to first-pass metabolism. To achieve therapeutic concentrations for systemic infections, innovative approaches like intravenous delivery or lipid-based nanocarriers may be necessary to enhance tissue penetration. Although the four-fold reduction in CBD MIC against S. epidermidis highlights the potentiating effect of DFNAC, the clinical relevance of this finding must be interpreted in the context of CBD pharmacokinetics. While such concentrations may be challenging to achieve through systemic administration, they are potentially attainable in topical, local, or biomaterial-based delivery systems. Consequently, the observed synergistic interaction may be particularly relevant for localised antimicrobial applications, including wound management, implant coatings, and surface-associated infections. Further pharmacokinetic and in vivo studies are required to determine whether the effective concentrations identified in vitro can be achieved and maintained at sites of infection.

Another question, however, revolves around the rising issue of whether the antibacterial effect stems from bactericidal or bacteriostatic actions. The ratio of minimum bactericidal concentration (MBC) to minimum inhibitory concentration (MIC) is essential for evaluating a formulation’s effectiveness against bacteria. A ratio of 4 or lower, with an MBC close to the MIC [51], indicates a strong bactericidal effect, meaning that the drug not only stops bacterial growth but also kills a majority of the bacteria [52]. These agents typically target critical cellular structures, such as the bacterial cell wall (e.g., penicillin and cephalosporins), or disrupt bacterial DNA, leading to cell death. This makes them crucial for treating severe infections like endocarditis and meningitis, particularly in immunocompromised patients [53]. Conversely, a bacteriostatic mechanism is indicated by a significantly higher MBC compared to the MIC. Here, the drug inhibits growth without killing the bacteria, allowing the immune system to clear the infection. Such agents interrupt reversible processes like protein synthesis (e.g., tetracycline) or folic acid metabolism (e.g., sulfonamides), and are effective for localised infections but less reliable for serious cases [54]. While the MBC/MIC ratio is a vital pharmacological tool, its significance can vary with bacterial strains and testing conditions. Additionally, a drug that is bacteriostatic against one species may be bactericidal against another, underscoring the complexity of these formulations in clinical use. The enhanced efficacy observed in S. epidermidis through synergistic interaction between DFNAC and CBD should, now, be interpreted as consistent with rather than proof of complementary actions from DFNAC and CBD leading to enhanced bacterial damage and growth inhibition. Previously published studies proposed CBD-induced alterations of bacterial membrane integrity that primarily disrupt bacterial membrane integrity [55,56], and DFNAC interferences with intracellular metabolic processes [42]. In-depth dedicated membrane permeability assays, together with additional molecular and biophysical approaches, will be required to confirm the mechanism underlying the observed antibacterial interaction between DFNAC and CBD.

Furthermore, previous studies also indicated pairing NSAIDs with antibiotics against S. epidermidis and S. aureus. DFNAC sodium against S. epidermidis indicated a high affinity for S. epidermidis proteins and TcaR enzymes and suggested molecular docking [45]. DFNAC combined with gentamicin against S. aureus showed a synergistic effect (FIC index 0.38) [15], suggesting efflux pump inhibition. Conversely, ketorolac combined with gentamicin against S. aureus biofilms demonstrated synergistic activity against S. aureus biofilms (FIC index 0.31), likely through modulation of the biofilm matrix [57].

Despite showing weak-to-moderate direct antibacterial activity (MICs ranging from 32 to 512 µg/mL), based on the checkerboard assays—the primary method for testing synergistic activity—NSAIDs exhibited synergistic effects with various antibiotics like gentamicin, ciprofloxacin, oxacillin, chloramphenicol, cefuroxime, and tetracycline (FIC indices generally < 0.5 to 0.625). Efflux pump inhibition is frequently proposed as an MOA [41]. Moving forward, more molecular studies are essential to validate these findings and explore the potential of NSAIDs as modulators or antibacterial adjuvants. However, challenges remain, such as achieving effective concentrations without toxicity and confirming mechanisms to ensure pharmacokinetic and pharmacodynamic compatibility and specificity and avoid off-target effects. Furthermore, our exploratory evidence indicates that the combination of DFNAC and CBD may become a significant antimicrobial pairing, aligning with the ongoing efforts to refine applications [58,59,60] and establish a regulatory framework [61] for future pharmacological products. It also aims to address methodological inconsistencies in standardised testing methods, culture media, and CBD sources.

Notably, we found no antibacterial activity against the Gram-negative strains Escherichia coli and Pseudomonas aeruginosa, which reflects their intrinsic resistance and highlights the combination’s inability to overcome the permeability barrier of their outer membranes. The intrinsic resistance of P. aeruginosa is well documented. It can be attributed to its highly impermeable outer membrane, efficient efflux pump systems, and adaptive and acquired resistance mechanisms [62], which collectively limit the penetration and activity of many antimicrobial compounds.

While it is widely accepted that CBD is highly effective against Gram-positive bacteria, including MRSA, there is a debate about its efficacy against Gram-negative bacteria. Many studies support the view that CBD is inactive against Gram-negative bacteria due to their protective outer membrane. However, a conflicting hypothesis suggests that certain Gram-negative bacteria—specifically Neisseria gonorrhoeae, Neisseria meningitidis, and Legionella pneumophila—are highly sensitive to CBD. This observation raises the question of whether CBD is a niche Gram-positive agent or a broad-spectrum candidate with tailored applications [27,34,55,63]. In the meantime, dose–response studies against E. coli and S. aureus indicated that DFNAC reduced metabolic activity without removing biofilm mass at therapeutic concentrations, resulting in significant reductions in colony-forming units within the biofilm as confirmed by Scanning Electron Microscopy [64]. However, the bactericidal effect against Gram-negative and Gram-positive bacteria—evident through inhibition of biofilm formation or reduction in metabolic viability—is limited at clinically relevant concentrations with standardised MBIC or MBEC values missing. The minimum bactericidal concentrations (MBCs) were consistently above 2000 µg/mL, indicating limited bactericidal potential [41]. Therefore, direct and consistent data on membrane disruption as well as in vivo or toxicity study results are still needed for conclusive mapping. To aid in drug formulation profiling, our study provided information on drug–drug interactions and confirmed the biocompatibility of the proposed combination of DFNAC and CBD. We highlighted the biocompatibility profile of the combination on human dermal fibroblasts (HDFs). Both MTT and LDH assays demonstrated that the antibacterial concentration (3.906 µg/mL) preserves cell viability and membrane integrity. Live/dead staining confirmed that most cells remained viable and exhibited normal morphology. The overall results indicate that the combination operates within a therapeutic window, achieving antimicrobial efficacy without inducing significant cytotoxicity. Moreover, the combination shows high biocompatibility. Investigations reported that CBD decreased the overall acidity levels in rats treated with DFNAC, suggesting that cannabidiol effectively treated the condition [65], or provided hepatoprotection against DFNAC sodium-induced hepatotoxicity [66]. However, the increasing interest in cannabis also requires a deeper understanding of the potential interactions between CBD and NSAIDs based on current knowledge and evidence [67,68].

Therefore, further rigorous quantitative biofilm assays, mechanistic membrane studies, and validation through clinical or animal models are essential before NSAIDs [42] and CBD [69] can be considered viable anti-biofilm adjuvants. Although DFNAC was tested in clinical trials involving patients with uncomplicated urinary tract infections and cellulitis [44], additional studies are necessary to complete its antimicrobial profile. Recent developments in plant-derived and synthetic cannabinoids [59] and NSAIDs [70] support the hypothesis that new therapeutic formulations [71,72] can be designed [73] specifically to address the antimicrobial crisis while meeting clinical applications [74] and regulatory requirements. Moreover, addressing antibiotic resistance through drug reprofiling will enhance formulations for personalised medicine.

Several limitations of the present study should be acknowledged. First, although the antibacterial activity of cannabidiol (CBD), diclofenac sodium (DFNAC), and their combination was evaluated against both reference ATCC strains and a limited number of clinical isolates, the number of clinical strains tested remains relatively small. While the observed antibacterial activity across both reference and clinical isolates supports the potential of the CBD-DFNAC combination, the variability observed among clinical isolates indicates that strain-specific susceptibility profiles may influence the magnitude of the interaction. Furthermore, while the observed antimicrobial and bactericidal effects across both reference and clinical isolates are encouraging, they may not fully capture the phenotypic and genotypic diversity encountered in clinical settings. Therefore, additional studies involving a larger collection of clinical isolates, including multidrug-resistant strains such as MRSA and resistant Staphylococcus epidermidis isolates, are required to validate the robustness, spectrum of activity, and translational potential of the CBD–DFNAC combination. Consequently, the present findings should be considered proof-of-concept evidence supporting further investigation toward clinical application.

Second, although checkerboard, MBC/MIC, and time-kill kinetic assays demonstrated enhanced antibacterial activity of the combination, the molecular mechanisms underlying this interaction were not directly investigated. The proposed mechanisms, including CBD-mediated membrane perturbation and DFNAC-associated interference with bacterial metabolic processes, are based on the previous literature and should therefore be considered hypotheses rather than experimentally confirmed mechanisms in the context of the present study. Additional investigations employing membrane permeability assays (e.g., SYTOX Green uptake or propidium iodide staining), transcriptomic analyses, proteomics, and metabolomics will be necessary to elucidate the molecular basis of the observed antibacterial effects.

Third, the study did not include experiments designed to evaluate the emergence of antimicrobial resistance during prolonged exposure to CBD, DFNAC, or their combination. Consequently, the potential of the combination to delay or prevent resistance development compared with monotherapy remains speculative and requires dedicated evolutionary and serial-passage studies.

Another limitation is the lack of activity observed against the Gram-negative strains Escherichia coli and Pseudomonas aeruginosa. While this finding is consistent with previous reports describing the intrinsic resistance of Gram-negative bacteria to cannabinoids due to the outer-membrane permeability barrier, the present study did not investigate whether membrane-permeabilising agents could restore susceptibility. Future studies should evaluate the DFNAC-CBD combination together with agents such as polymyxin B or colistin to determine whether the antibacterial spectrum can be expanded toward Gram-negative pathogens. In addition, a dedicated Gram-negative antibiotic control was not included in the original experimental design, limiting direct benchmarking of antibacterial efficacy against established therapies.

An additional limitation of the present study concerns the concentration range evaluated in the cytocompatibility assays. Cell viability was assessed using concentrations encompassing the antibacterial MIC and extending up to 32 µg/mL, which represents approximately an eight-fold increase over the effective antimicrobial concentration. While this range was selected to evaluate the safety margin associated with biologically relevant antibacterial doses, it does not establish the complete toxicological profile of the formulation. Future investigations should include substantially higher concentrations, prolonged exposure periods, additional primary human cell types, and in vivo biocompatibility studies to define the maximum tolerated dose and therapeutic window more comprehensively.

Finally, the current findings are based exclusively on in vitro experiments. The pharmacokinetic behaviour, tissue distribution, stability, and in vivo antibacterial efficacy of the DFNAC CBD combination remain unknown. Although the concentrations identified in vitro may be difficult to achieve through systemic administration, they may be attainable in localised applications such as wound dressings, biomaterial coatings, implant-associated infections, and topical formulations. Therefore, future studies should focus on formulation development, pharmacokinetic characterisation, and validation in relevant animal and non-animal models before clinical translation can be considered.

Taken together, the present findings provide proof-of-concept evidence supporting the antibacterial potential of the DFNAC-CBD combination against Gram-positive staphylococci. The pronounced synergistic interaction observed against S. epidermidis, compared with the additive effect detected in S. aureus, may reflect intrinsic biological differences between these species. Although both are Gram-positive staphylococci, they differ in membrane composition, regulatory pathways, biofilm-associated proteins, and stress-response mechanisms. CBD is believed to primarily affect membrane integrity, whereas diclofenac may interfere with intracellular metabolic processes and bacterial regulatory networks. The greater susceptibility of S. epidermidis to the combined action of these compounds may explain the stronger reduction in MIC observed in this species. In contrast, the more complex adaptive responses of S. aureus may attenuate the potentiating effect of the combination, resulting in an additive rather than synergistic interaction. Further mechanistic investigations will be necessary to clarify the molecular basis of these species-specific responses. Also, additional pharmacological and translational studies are required to fully establish complex clinical perspectives.