In general, all of the hemp ethanol extracts showed some degree of in vitro antibacterial activity against all of the bacterial strains tested, including both the antibiotic-resistant and antibiotic-sensitive forms; however, their potency varied substantially from 128 μg/mL to 1024 μg/mL. While these MIC values are higher than those reported in the literature for individual cannabinoids, our results are partially consistent with those obtained in recent studies demonstrating the growth-inhibitory effects of crude cannabis extracts against staphylococcal strains over a wide MIC range from 8 to 8552 µg/mL [19,20,21,22]. Specifically, lower values were reported by Skala et al. (2022), who reported an MIC of 8 μg/mL for an ethanolic cannabis extract against S. aureus (ATCC 29213) [22]. Additionally, the MICs for the seed (25 μg/mL) and whole plant (50 μg/mL) for the ethanol extracts were measured via the microdilution broth method [19]. Although the MIC value reported by Ali et al. (2012) is significantly lower than that from our findings, it cannot be fully compared with our findings because the cannabinoid content of the plant was not observed [2]. In contrast, in the study by Skala et al. (2022) [22], the total cannabinoid content of the two ethanol extracts studied was higher (245,940 and 329,100 µg/g) than that in our study (Figure 1). However, the total content cannot be fully compared due to the different amounts of cannabinoids analysed (7 tested extracts vs. 14 tested extracts in our study). However, the amounts of individual cannabinoids contained in the extracts were greater (80–261,750 µg/g) than those in our study (0–15,208.79 µg/g). In addition, our extracts did not contain as much Δ⁹-THC (40.61–513.27 µg/g), CBC (105.28–301.71 µg/g) or CBG (17.12–1163.19 µg/g) as the extracts described in Skala et al. (2022) [22] did, where the values ranged from 35,240 to 51,230 µg/g); 1350–1450 µg/g and 540–1050 µg/g for Δ⁹-THC, CBC and CBG, respectively; this could explain the lower MICs reported by Skala et al. (2022) [22], as the antibacterial activity of these individual cannabinoids has been well studied [11]. However, the antibacterial activity was determined for whole extracts containing different amounts and contents of cannabinoids and other substances that interact with each other. In addition, Skala et al. (2022) used a medicinal cannabis strain containing relatively high amounts of cannabinoids [22]. Our study used industrial hemp, which generally contains lower amounts of cannabinoids but is permitted for use in livestock under legislation (0.2% THC content [23]), and, thus, could be used in this area of research in subsequent in vivo experiments. Giselle et al. (2023) reported values comparable to our findings for S. aureus (ATCC 29213), in which the MIC of an inflorescence extract was 115.25 μg/mL, and the MIC of a root extract was 8552.38 μg/mL [21]. Additionally, Kaur et al. (2015) determined the MIC of a methanolic extract of C. sativa to be 1560 μg/mL via the agar well diffusion method [20].

In streptococci, the antibacterial activity of specific cannabinoids was investigated in strains of Streptococcus mutans (MIC 2.5 μg/mL), Streptococcus sanguis (MIC 1 μg/mL), Streptococcus sobrinus (MIC 5 μg/mL), Streptococcus salivarius (MIC 5 μg/mL) [24], Streptococcus pyogenes (2–5 μg/mL), Streptococcus milleri (1–2 μg/mL) [25] and Streptococcus pneumoniae (1–2 μg/mL) [26]. The values reported in this study are greater than those reported in the literature, which has focused on assessing the activity of individual cannabinoids present in C. sativa, probably because samples from whole C. sativa extracts may be expected to display interactions among the substances present or different mechanisms of action. The MIC values in our study are similar to those of ethanol extracts investigated against Str. pyogenes (128 μg/mL) by Skala et al. (2022) [22].

The cannabinoid content of C. sativa depends on many biotic and abiotic factors such as nutrient availability, growth cycle stage, storage, light intensity and ambient temperature [27]. Differences in the cannabinoid content were observed among the five extracts tested, which may influence their antibacterial activity; this is also indicated by our results, in which higher antibacterial activity was observed in extracts with higher cannabinoid contents (Bialobrzeskie, mixed and Santhica 27) and lower antibacterial activity was observed in extracts with lower cannabinoid contents (Felina 32 and Futura 75). These results partially correlate with the different antibacterial activities of the flower and root extracts. The antibacterial activity of cannabinoids in flowers has been demonstrated at lower MICs (1–16 μg/mL) [14] and (MIC 115.25 μg/mL). Meanwhile, in the root extracts, a relatively high MIC was detected, and at the same time, the absence of cannabinoids was noted (MIC 8552.38 μg/mL) [21]. These findings suggest that the cannabinoid content could affect the antibacterial activity of cannabis. However, it should be noted that the antibacterial activity cannot be attributed solely to the cannabinoids themselves but rather to the synergy of substances contained in cannabis in general [21].

To our knowledge, this study is the first to compare the antibacterial effects of ethanol extracts from single hemp cultivars against both antibiotic-resistant and antibiotic-sensitive clinical and standard S. aureus, Str. agalactiae and Str. dysgalactiae strains.