A series of experiments were conducted to evaluate the effects of ergot alkaloids (dihydroergotamine, ergonovine, and ergotamine) on E. coli O157:H7 in both pure and mixed ruminal fluid culture. Alkaloids were added to solutions of E. coli O157:H7 strains 933 (pure and ruminal cultures) and 6058 (ruminal culture only), and growth rates and colony-forming units (CFU) of E. coli O157:H7 were measured. Two mixtures of all three alkaloids at either 2 or 500 microM for each alkaloid decreased (p < 0.001) the growth rate of E. coli O157:H7 in pure culture compared to the individual alkaloids. Dihydroergotamine tended (p = 0.07) to reduce growth rate of E. coli O157:H7 in pure culture compared with ergonovine or ergotamine alone. Increased concentrations of dihydroergotamine and ergotamine decreased (p < 0.003) growth rate of E. coli O157:H7 but increasing concentrations of ergonovine did not influence (p > 0.10) E. coli O157:H7 growth rate. Similar to results in pure culture, a mixture of all three alkaloids at various concentrations for each alkaloid decreased (p < 0.001) the CFU of E. coli O157:H7 strain 6058 in mixed ruminal culture compared to the individual ergot alkaloids. Dihydroergotamine decreased (p = 0.04) CFU of E. coli O157:H7 strain 6058 when compared to ergonovine but CFU were similar (p > 0.10) between dihydroergotamine and ergotamine. Ruminal and (or) intestinal populations of E. coli O157:H7 may be influenced in livestock consuming endophyte-infected tall fescue, and these alterations could be due to the presence of ergot alkaloids in fescue plants.