Manure from draft animals deposited in fields during vegetable and fruit production may serve as a potential source of preharvest pathogen contamination of foods. To better quantify this risk, we determined the prevalence of Escherichia coli O157:H7 in horses. Between June and September 2009, freshly voided fecal samples were collected from horses stabled on 242 separate premises in Ohio, USA. Overall, the prevalence of E. coli O157:H7 was 1 of 242 (0.4% prevalence, 95% confidence interval [CI] = 0.01 to 2.28). E. coli O157:H7 was recovered from none of the 107 equine fecal samples (0% prevalence, 95% CI = 0.00 to 3.39) that originated from locations without ruminant presence, and only 1 of the 135 horse fecal samples (0.7% prevalence, 95% CI = 0.02 to 4.06) from sites where ruminants were also present. The lone positive sample was collected from a horse that was costabled with a goat. Subsequent sampling at that location identified indistinguishable subtypes of E. coli O157:H7 present in the cohoused goat, in the environment, insects, sheep, and other goats housed in an adjacent field. E. coli O157:H7 was not isolated from the five subsequent samples from this horse. These data indicate that E. coli O157:H7 carriage by horses is an uncommon event.