We sampled 325 small and medium-sized wild mammals in Ontario, Canada in 2007 and 2010 to determine the prevalence and characteristics of Clostridium difficile in wild mammals living in proximity to captive wildlife and livestock. Clostridium difficile was isolated from five of 109 animals (4.6%) on four of 25 farms (16%), but was not isolated from any of the 216 samples from raccoons (Procyon lotor) living on the grounds of the Toronto Zoo. The positive animals included two raccoons from one beef farm, one raccoon from a different beef farm, one raccoon from a swine farm, and a shrew (Blarina brevicauda) from a dairy farm. None had evidence of gastrointestinal disease. Three of the five isolates were toxinotype variants (II, IV, and XIII) that are rarely identified in humans and domestic animals. The other two were toxinotype 0, a common toxinotype in humans and animals; however, all five isolates were of different ribotypes. None of the recovered ribotypes were recognized as ribotypes present in the authors’ reference library of over 3,000 human and domestic animal C. difficile isolates. Neither the public health nor the animal health relevance of these findings is clear. It is not known whether C. difficile is a pathogen of small and medium-sized wild mammals, although the susceptibility of various laboratory species suggests it could cause disease.