The relationship between the gut microbiota and Clostridioides difficile, and its role in the severity of C.difficile infection in humans is an area of active research. Intestinal carriage of toxigenic and non-toxigenic C.difficile strains, with and without clinical signs, is reported in animals, however few studies have looked at the risk factors associated with C.difficile carriage and the role of the host gut microbiota. Here, we isolated and characterized C.difficile strains from different animal species (predominantly canines (dogs), felines (cats), and equines (horses)) that were brought in for tertiary care at North Carolina State University Veterinary Hospital. C.difficile strains were characterized by toxin gene profiling, fluorescent PCR ribotyping, and antimicrobial susceptibility testing. 16S rRNA gene sequencing was done on animal feces to investigate the relationship between the presence of C.difficile and the gut microbiota in different hosts. Here, we show that C.difficile was recovered from 20.9% of samples (42/201), which included 33 canines, 2 felines, and 7 equines. Over 69% (29/42) of the isolates were toxigenic and belonged to 14 different ribotypes including ones known to cause CDI in humans. The presence of C.difficile results in a shift in the fecal microbial community structure in both canines and equines. Commensal Clostridium hiranonis was negatively associated with C.difficile in canines. Further experimentation showed a clear antagonistic relationship between the two strains invitro, suggesting that commensal Clostridia might play a role in colonization resistance against C.difficile in different hosts.