Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.