Abstract: The purpose of the present study was to evaluate the in vitro antibacterial activity of rifaximin against the common bacterial pathogens associated with bovine mastitis. Agar dilution method was used to determine the minimum inhibitory concentration（MIC）of rifaximin against clinical isolates of E. coli, Streptococci and Staphylococci from dairy cows with mastitis.The MIC range of rifaximin against 19 E. coli strains, 25 Staphylococcus strains and 18 Streptococcus strains were 8-16 μg/m L,0.002-0.06 μg/m L and 0.06-2 μg/m L, respectively. The MIC of the most clinical isolates was similar with that of their corresponding standard strains, indicating that they were sensitive to rifaximin. The combined data suggest that rifaximin exhibits strong antibacterial activity against the common bacterial pathogens associated with bovine mastitis.