Clostridium difficile (C. difficile) is one of the main causes of diarrhea and enterocolitis in horses. The goal of this study was to investigate the prevalence and risk factors associated with C. difficile infections in adult horses and foals with and without diarrhoea. Fresh faecal samples were collected from 407 horses originating from 35 stables in Eastern province, Saudi Arabia. The collected samples were cultured to isolate C. difficile. ELISA was used for detection of C. difficile toxins (A and B). Intestinal and cecal samples were collected from two dead horses for histopathological examination. Histopathological findings were mainly coagulative necrosis of the mucosa and marked mucosal and submucosal thrombosis. Hemorrhages, fibrinous exudate and leukocytic infiltration were seen throughout the mucosa and submucosa. Focal mucosal ulcerations and fibrinonecrotic pseudomembranes were also observed. C. difficile were detected in a total of 24 (5.9%) horses and its toxins were detected in 13 (54%) isolates. Toxigenic C. difficile infections (positive cases) were closely associated with diarrhetic foals than normal adult horses. Furthermore, there was close association between toxigenic C. difficile infections and signs of colic and bloody faeces. The frequency of positive cases was higher in horses under antibiotic therapy than those without treatment. The study concludes that acute colitis in adult horses and diarrhea in foals previously treated with antibiotics is associated C. difficile infection.