Antibiotic resistance in staphylococcal strains and its impact on public health and agriculture are global problems. The development of new anti-staphylococcal agents is an effective strategy for addressing the increasing incidence of bacterial resistance. In this study, ethanolic extracts of Cannabis sativa L. made from plant parts harvested during the whole vegetation cycle under various nutritional treatments were assessed for in vitro anti-staphylococcal effects. The results showed that all the cannabis extracts tested exhibited a certain degree of growth inhibition against bacterial strains of Staphylococcus aureus, including antibiotic-resistant and antibiotic-sensitive forms. The highest antibacterial activity of the extracts was observed from the 5th to the 13th week of plant growth across all the nutritional treatments tested, with minimum inhibitory concentrations ranging from 32 to 64 µg/mL. Using HPLC, Δ9-tetrahydrocannabinolic acid (THCA) was identified as the most abundant cannabinoid in the ethanolic extracts. A homolog of THCA, tetrahydrocannabivarinic acid (THCVA), reduced bacterial growth by 74%. These findings suggest that the cannabis extracts tested in this study can be used for the development of new anti-staphylococcal compounds with improved efficacy.