Aims: To examine the effects of ionophores on Salmonella and Escherichia coli O157:H7 in pure and mixed ruminal fluid cultures. Methods and Results: Four Salmonella serotypes (Dublin, Derby, Typhimurium, and Enteriditis) and two strains of E. coli O157:H7 (ATCC 43895 and FDIU 6058) were cultured in the presence of varying concentrations of ionophores (monensin, lasalocid, laidlomycin propionate, and bambermycin) in pure and mixed ruminal fluid cultures. Bacterial growth rates in pure culture were not affected (P>0.10) by ionophores at concentrations up to 10 times the approximate rumen ionophore concentration under normal feeding regimens. Likewise, ionophores had no effect (P>0.10) on Salmonella or E. coli CFU plated from 24-h ruminal fluid incubations. Ionophore treatment decreased (P<0.01) the acetate : propionate ratio in ruminal fluid cultures as expected. Conclusions: Ionophores had no effect on the foodborne pathogens Salmonella and E. coli O157:H7 in vitro . Significance and Impact of the Study: The results suggest that ionophore feeding would have little or no effect on Salmonella or E. coli populations in the ruminant.