The agar diffusion assay revealed differences in growth inhibition among the tested bacterial strains and EO types (Figure 7). Two-way ANOVA showed a significant effect of EO type (F = 441.6, p < 0.001), bacterial strain (F = 35.3, p < 0.001), and their interaction (F = 9.4, p < 0.001) on inhibition zone diameter. Because the diffusion of EO constituents through agar depends on their physicochemical properties, inhibition zone diameters should be regarded as preliminary indicators of antibacterial activity rather than direct measures of antibacterial potency.

The antimicrobial activity of the tested hemp EOs varied markedly with EO type and bacterial strain. The highest activity was observed for infructescence-derived EOs, particularly LFR, which inhibited the growth of most tested strains at concentrations ranging from 0.025 to 0.20 mg mL⁻¹ (Table 3). In contrast, inflorescence-derived EOs generally exhibited higher MIC values, with no inhibition detected within the tested concentration range for several strain–EO combinations.

Gram-positive bacteria were generally the most susceptible to the infructescence-derived EOs, especially Staphylococcus epidermidis (MIC 0.025–0.050 mg mL⁻¹), Staphylococcus aureus (0.05–0.10 mg mL⁻¹), and acne-associated bacteria (0.025–0.10 mg mL⁻¹). LFR and UFR were also active against the oral streptococci Streptococcus mutans and Streptococcus sanguinis, with MIC values of 0.05–0.10 mg mL⁻¹, whereas the corresponding MICs for inflorescence-derived EOs were up to 16–32-fold higher or exceeded the highest tested concentration. Although Gram-negative bacteria were generally less susceptible to the tested EOs, those obtained from UFR and LFR inhibited both E. coli and P. aeruginosa (MIC 0.20–0.40 mg mL⁻¹), indicating a relatively broad spectrum of antimicrobial activity. It should be highlighted that, for several strains, the MIC values of the inflorescence-derived EOs exceeded the highest tested concentration (1.60 mg mL⁻¹), suggesting that the observed superiority of the infructescence-derived EOs may have been underestimated. It is also noteworthy that antimicrobial efficacy appeared to be more strongly influenced by the type of EO than by the taxonomic affiliation of the bacterial strains, with UFR and particularly LFR showing greater activity against most tested strains. Sparfloxacin, used as the reference antimicrobial agent, exhibited markedly lower MIC values than the tested EOs, confirming the validity of the assay.

Principal component analysis (PCA) was used to explore associations between the chemical composition of the hemp EOs and bacterial growth inhibition zones (Figure 8). The first three axes (PCs) explained 97.42% of the total variance (76.03% for Axis 1, 13.41% for Axis 2, and 7.98% for Axis 3) (Table 4). PC1 primarily differentiated EOs associated with higher contents of α-Pinene, Myrcene, and (E)-Caryophyllene from those associated with higher contents of (Z)-Caryophyllene and (E)-β-Farnesene. Growth inhibition zones for all tested bacterial strains loaded positively on PC1, indicating that greater antibacterial activity was associated with the former compositional profile. This association was particularly strong for PA, EF2, PAC2, and SS, whereas EF1, SA, and SE showed relatively greater contributions to Axis 2. PC2 was mainly associated with Caryophyllene oxide and differentiated the responses of SA, SE, EF1, and CA from those of EC, PAC2, and SS. PC3 was primarily associated with CBD and further differentiated the responses of EC, SM, PAC1, and SS from those of EF1 and SA. Overall, the PCA revealed a clear association between EO composition and antibacterial activity: the broader activity of UFR and particularly LFR was associated with higher proportions of α-Pinene, Myrcene, and (E)-Caryophyllene, whereas the more selective activity of UFL was associated with a distinct compositional profile, including higher proportions of (Z)-Caryophyllene and Caryophyllene oxide. These multivariate associations should not, however, be interpreted as evidence of a causal contribution of individual EO constituents to antibacterial activity.

Previous studies have demonstrated antimicrobial activity of hemp EOs, although the investigated microorganisms and EO profiles varied considerably among studies. Zengin et al. [23] reported activity of EOs from Futura 75 against S. aureus ATCC 29213 and clinical S. aureus isolates, including effects assessed by MIC, MBC, and antibiofilm analyses. A broader screening of fibre-type hemp EOs, including Futura 75, demonstrated activity against several Gram-positive bacteria, including S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, Listeria spp. and Bacillus spp. [12]. Similarly, comparative analyses of Carmagnola, Fibranova, and Futura EOs against Gram-positive and Gram-negative bacteria and yeasts identified Futura as the most active of the three cultivars [11]. These studies provide a useful context for the present results while also highlighting the substantial influence of cultivar, EO composition, and bacterial strain on the observed antibacterial activity.

The present study extends the available evidence on the antibacterial activity of Futura 75 EOs to bacterial strains that have received limited attention in previous studies. The strongest activity was observed for EO obtained from LFR, which produced the highest inhibition zones for almost all tested strains, including S. aureus ATCC 25923, S. epidermidis ATCC 12228 and E. faecalis PCM 896 (14.67, 15.67, and 15.43 mm, respectively). Although Staphylococcus spp. and Enterococcus spp. have previously been included in studies on hemp EOs’ antibacterial activity, strain-specific data for Futura 75 remain limited. Šovljanski et al. [22] tested S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 using EO from industrial hemp cv. Helena. Therefore, the present results extend the strain-specific evidence available for the antibacterial activity of Futura 75 EOs.

The anti-staphylococcal activity observed in the present study is consistent with previous reports, although its magnitude varied markedly among the tested EOs. Zengin et al. [23] reported MIC and MBC values of 8 and 16 mg mL⁻¹, respectively, for Futura 75 EOs against S. aureus, whereas Iseppi et al. [12] found substantial variation in anti-staphylococcal activity among EOs from different hemp cultivars. In the present study, UFR and particularly LFR showed considerably lower MIC values against S. aureus and S. epidermidis than UFL and LFL. Although direct quantitative comparisons among studies should be made cautiously because of differences in EO composition and experimental conditions, these findings further indicate that anti-staphylococcal activity is strongly influenced by the chemical profile of the EO.

The results obtained for E. faecalis PCM 896 further support the sensitivity of enterococci to hemp EOs. Antibacterial activity against Enterococcus hirae, E. faecium, and E. faecalis has been reported for EOs from different hemp cultivars [11,12], while Šovljanski et al. [22] identified E. faecalis as one of the most susceptible bacteria to industrial hemp EO. In the present study, EOs obtained from LFR, UFR and UFL exhibited high activity against E. faecalis PCM 896, whereas the EO obtained from LFL was considerably less active. These findings support the susceptibility of selected Enterococcus strains to hemp EOs, while further indicating that the magnitude of the antibacterial efficacy depends strongly on EO composition.

A particularly important finding of the present study is the activity of hemp EOs against Gram-negative bacteria. Earlier studies often reported lower susceptibility of Gram-negative bacteria to hemp EOs, which is commonly attributed to the outer membrane barrier. Consistent with this general pattern, Šovljanski et al. [22] found no activity of EO from industrial hemp cv. Helena against Gram-negative bacteria, yeasts or fungi. In contrast, Zheljazkov et al. [72] reported strong antimicrobial activity of selected hemp chemovar EOs against several Gram-negative and foodborne bacteria (including E. coli, Salmonella enterica and Listeria monocytogenes), whereas Nafis et al. [73] demonstrated inhibition of E. coli and P. aeruginosa by Moroccan hemp EO, with MIC values of 1.2 mg mL⁻¹ for both strains. In this context, the activity of Futura 75 EOs obtained from LFR and UFR against E. coli ATCC 25922 and P. aeruginosa ATCC 27853 is particularly noteworthy. The activity against P. aeruginosa ATCC 27853 is especially interesting because the same strain was resistant to the hemp EO tested by Šovljanski et al. [22]. Together, these contrasting findings indicate that the susceptibility of Gram-negative bacteria to hemp EOs may vary substantially depending on EO composition and other experimental factors.

Relatively few studies have investigated the activity of industrial hemp EOs against acne-associated bacteria [11,12,18,21,22,23,47,72,73,74]. In the present study, EOs obtained from LFR showed pronounced activity against the tested acne-associated strains, producing inhibition zones of 13.00 mm against C. acnes ATCC 11827 and 13.67 mm against C. acnes PCM 2334. EOs obtained from both LFR and UFR also inhibited C. acnes PCM 2400, with inhibition zones of 13.00 mm. These findings extend the available evidence on the antibacterial spectrum of Futura 75 EOs to acne-associated bacterial strains. However, their relevance to potential dermatological applications requires further investigation using complementary approaches, including bactericidal, time-kill, and antibiofilm assays, as well as appropriate skin-related experimental models.

The activity of the tested hemp EOs against oral streptococci represents another relevant finding of the present study. Essential oil obtained from LFR inhibited S. mutans PCM 2020 and S. sanguinis PCM 2335, producing inhibition zones of 14.00 and 13.67 mm, respectively. Nissen et al. [11] previously evaluated hemp EOs against another streptococcal species, Streptococcus salivarius subsp. thermophilus, whereas evidence concerning the activity of Futura 75 EOs against oral streptococci remains limited. The present findings therefore extend the antibacterial spectrum demonstrated for Futura 75 EOs to the tested oral streptococci and provide a rationale for further studies addressing cariogenic biofilms, bacterial adhesion, and interactions within multispecies oral microbiota.

The variability observed among the studied EOs is consistent with previous reports showing substantial cultivar- and composition-dependent differences in the antibacterial activity of hemp EOs. Novak et al. [74] showed considerable variation in EO composition among hemp cultivars accompanied by generally modest and cultivar-dependent antimicrobial activity. Similarly, substantial differences in the proportions of major sesquiterpenes have been reported among novel Serbian hemp chemovars [72], while Palmieri et al. [18] demonstrated that both cultivar and distillation time may influence EO composition and antimicrobial activity. Importantly, antibacterial effects cannot necessarily be attributed to individual major constituents. Pieracci et al. [45] showed that hemp EOs were more active than their main constituent, β-Caryophyllene, tested individually, supporting a contribution of interactions among EO constituents. Consistently, Pellegrini et al. [21] associated the antimicrobial activity of Futura 75 EO with several major terpenes, including β-Caryophyllene, Myrcene, Humulene and α-Pinene, while also suggesting a contribution of minor constituents. Thus, the antibacterial activity of hemp EOs is more appropriately interpreted in relation to their overall chemical profile and potential interactions among constituents than to the abundance of a single compound.

The higher proportion of monoterpenes relative to sesquiterpenes contributes to a lighter and more volatile chemical profile of the EO. Owing to their smaller molecular size and physicochemical properties, monoterpenes may interact differently with biological membranes than the heavier sesquiterpenes, which may contribute to differences in the biological activity of EOs [75,76]. In contrast, β-Caryophyllene, a bicyclic sesquiterpene, demonstrates selective CB2 receptor agonism attributed to its rigid molecular structure, which enables interaction with cannabinoid receptors while avoiding CB1-mediated psychoactive effects [77]. However, the biological activity of complex EOs cannot be directly predicted from the abundance or properties of individual constituents, as interactions among multiple components may substantially influence their overall effects.

Taken together, these findings suggest that the biological activity of hemp EOs is determined not only by the presence of individual monoterpenes, sesquiterpenes, and cannabinoids, but also by their relative proportions and potential synergistic interactions. Most importantly, the present study demonstrates that bracts and leaves remaining after seed harvesting, which represent an underutilized post-harvest biomass, can serve as a source of biologically active EOs. The observed differences between cytotoxic effects toward mammalian cells and antibacterial activity further indicate that greater cytotoxicity does not necessarily correspond to greater antibacterial effect. In the present study, the inflorescence-derived UFL and LFL oils, which were dominated by sesquiterpenes and contained substantially higher proportions of (Z)-Caryophyllene, showed greater cytotoxic and haemolytic effects but generally lower antibacterial activity than the infructescence-derived UFR and LFR oils. This apparent difference may reflect the fundamentally different organisation and physicochemical properties of mammalian plasma membranes and bacterial cell envelopes. In bacteria, the peptidoglycan layer and, in Gram-negative species, the additional outer membrane may restrict the accessibility of EO constituents to the cytoplasmic membrane, while differences in membrane lipid composition and surface properties may further influence susceptibility. In addition, interactions among EO constituents may result in different effects on mammalian and bacterial membranes [78,79,80]. Thus, the contrasting cytotoxic and antibacterial profiles of the tested EOs are likely to reflect their overall chemical composition and the distinct structural properties of their cellular targets rather than the abundance of a single constituent. The combination of antibacterial activity with comparatively low cytotoxicity and haemolytic activity observed for UFR and particularly LFR supports further investigation of infructescence-derived EOs in application-oriented antimicrobial studies.