Clostridium difficile is considered one of the most important causes of enterocolitis associated with diarrhea in horses especially foals that may lead to necrohaemorrhagic enterocolitis with high mortality rate. Diarrhea mostly developed while treating with antibiotics, however, it may develop without any history of antibiotics treatment. In this study, twenty faecal samples and seven rectal swabs from foals suffering from diarrhea were collected and cultured onto Cefoxitin Fructose Agar (CFA) as specific growth medium. Isolates from twenty-one (77.7%) of these samples gave the characteristic microscopical examination and colony morphology as well as they gave negative results with catalase and oxidase tests. Also, they gave fluorescence after exposure to long wave length ultraviolet light. Specific agglutination kit was used for confirmation, revealed nineteen isolates (70.4%) gave positive for agglutination. The A and B crude toxins were prepared from the isolates, toxogencity test in mice was done for the prepared toxins as preliminary test for toxogencity.Nine (33.3%) isolates found to be lethal to mice, then SDS-PAGE was done to the fifth isolate (as it gave the highest MLD, indicating its the most toxogenic isolate) where the toxins gave bands at molecular masses of 308 and 270 kDa, which  resemble that of toxins A and B of Clostridium difficile, respectively. Finally, the isolated Clostridium difficile recommended to be used in preparation of vaccine for controlling the severity of enterocolitis disease among horses.