Kefir beverage is a complex probiotic, from a microbial symbiotic complex that is obtained mainly from cow's milk fermentation by kefir grains. Some people do not consume cow's milk. Plant-based milk can be alternative for animal milk. The purpose of this study is to investigate the antibacterial activity of kefir samples prepared with cow's milk and almond milk. Activated kefir grains were added to cow's milk (full-fat and low-fat) and almond milk (with and without sucrose) and the fermentation process was performed at 25 and 37°C. Grains were separated from kefir extract, and the antibacterial activity of kefir extract against 8 bacteria was evaluated by the well method. Fermentation temperature had a significant effect on the antibacterial activity of kefir samples prepared with cow's milk in all tested bacteria except for E. faecalis (P<0.05) and kefir samples prepared at 37°C compared to 25°C showed more antibacterial activity. The type of cow's milk (full-fat and low-fat) did not show a significant effect on the antibacterial activity against the tested bacteria, except for S. aureus, S. typhimurium, and B. cereus. E. faecalis and S. typhimurium were the most sensitive and resistant bacteria, respectively, to kefir samples prepared with cow's milk. Despite the lack of effect of fermentation temperature on the antibacterial activity of kefir samples prepared with almond milk, the presence of sucrose showed a significant effect on the antibacterial activity against E. faecalis, S. dysenteriae, B. cereus and P. aeruginosa (P<0.05), which indicates the positive effect of the presence of sucrose on the antibacterial activity. In general, fermentation at temperature of 37°C is recommended to achieve the highest antibacterial activity of kefir samples prepared with cow's milk, as well as adding sucrose to almond milk to achieve the highest antibacterial activity against E. faecalis, S. dysenteriae, B. cereus and P. aeruginosa.