Background: Calf diarrhea is an important disease that is caused by different pathogens including bacteria, virus and parasites and is associated with economic losses. Objectives: In this study, we evaluated the antibacterial activities of Zatacin (Z. multiflora aerial part ethanol extract), colistin, co-Trimoxazole and enrofloxacin against clinical isolates of Salmonella sp, E. coli and Campylobacter sp isolated from diarrheic calves. Methods: Disc diffusion method and microbroth dilution assay were used for antimicrobial evaluation. Results: In disc diffusion method, the antibacterial activity of Zatacin increased dose dependently. The sensitivity of different isolates of E. coli, Salmonella sp and Campylobacter sp to Zatacin was almost the same. The antibacterial activity of Zatacin was lower than that of enrofloxacin but it was higher than co-Trimoxazole and colistin. The means of MIC values of Zatacin for E.coli were higher than that of Campylobacter sp and Salmonella sp but its means of MBC values for E. coli were lower than that of two other bacteria. Conclusions: Zatacin can be used as an antimicrobial agent in treatment of infectious causes of calf scours instead of antibiotics with undesired adverse effects on animal and humans.