Although progress has been made in the last decade in understanding the causes of colitis in horses, perhaps 60% of cases of fatal colitis in horses have no known cause. The role of type A Clostridium perfringens strains was evaluated in this study. Fecal samples from 55 horses (43 adults, 12 foals) with colitis were cultured for Clostridium difficile, Salmonella, and C. perfringens. Feces were also tested for C. difficile toxins A/B and C. perfringens toxins (alpha [CPA], beta2 [CPB2], enterotoxin [CPE]) using enzyme-linked immunosorbent assays (EIA). All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. No CPE was detected but 36.4% and 18.2% of animals were positive for CPA and CPB2 toxin, respectively. Subsequently, five C. perfringens isolates per fecal sample were genotyped and the supernatants of each of these isolates were evaluated for toxicity. None of the isolates were cpe, netB or tpeL positive, but atypical cpb2 and consensus cpb2 were identified in 13.6% and 3.6% isolates, respectively. All equine C. perfringens isolates showed mild toxicity effects compared to CPB producing C. perfringens positive control. Based on this study population, there was no evidence that C. perfringens had an important role in equine colitis.