Diseases caused by staphylococci and streptococci are a serious burden on livestock production, causing significant losses. In addition, the associated antibiotic resistance of these pathogens often makes treatment impossible or prolonged. Cannabis sativa L. contains many compounds with antibacterial properties and shows great potential as a natural antimicrobial agent for agricultural use against both of these bacterial species. The aim of this study was to compare the in vitro antibacterial activity of ethanol extracts from five cultivars of hemp, namely, Bialobrzeskie, Felina 32, Futura 75, mixed and Santhica 27, against Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae. All five cultivars exhibited a certain degree of inhibitory effect against all the pathogens tested with minimum inhibitory concentrations (MICs) ranging from 128 to 2048 μg/mL. The extract from the Santhica 27 cultivar was the most effective antibacterial agent with the lowest MIC value of 128 μg/mL against Str. agalactiae and two clinical isolates of S. aureus, followed by Bialobrzeskie and mixed cultivars with the same growth-inhibitory potential against Str. agalactiae. The extracts from the Felina 32 and Futura 75 cultivars presented only weak activity with MIC values ranging from 256 to 2048 μg/mL. The extract from the Santhica 27 cultivar appears to be a promising product for future use in the treatment of staphylococcal and streptococcal infections in livestock.