During a 24 month period from 2007 to 2009, 174 faecal specimens from horses in Australia (predominantly from Western Australia) were tested for Clostridium difficile. C. difficile was isolated from 14 (23 %) of 62 diarrhoeal animals (including 10 foals) and from none of 112 healthy adult horses. These isolates were toxin profiled by PCR for toxin A, toxin B and binary toxin, and ribotyped. Ten of the equine isolates were A(+)B(+) CDT-. Other toxin profiles detected were A(-)B(-)CDT(+) (one isolate), A(+)B(+)CDT(+) (two isolates) and A(-)B(-)CDT(-) (three isolates). There were six different ribotypes detected in the horses, ribotype 012 being the most common with six isolates. Two horses (one adult and one foal) had two strains of C. difficile isolated on different days. These strains had the same toxin profile but different ribotypes. None of the equine isolates was ribotype 078, which is A(+)B(+)CDT(+) and a significant cause of animal disease overseas. All isolates were susceptible to metronidazole and vancomycin. These results suggest that the epidemiology of C. difficile in horses in Australia is currently similar to that in other parts of the world, but requires further surveillance to monitor changes.