© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.Objectives: Increasing reports of genetic overlap between animal and human sources of Clostridium difficile necessitate an understanding of antimicrobial susceptibility and resistance in these populations. In this study, we sought to investigate the in vitro activities of 13 antimicrobials against a unique collection of clade 5 C. difficile isolates of Australian animal and human origin. Methods: The collection comprised 171 C. difficile strains of human (n = 91) and animal (n = 80) origin in Australia in the last decade. The collection encompassed seven different clade 5 PCR ribotypes (RTs; 033, 078, 126, 127, 237, 281 and 288), seven STs and three toxin gene profiles. MICs were determined by agar incorporation methodology. Results: All isolates were fully susceptible, with little variation between strain populations, to vancomycin, metronidazole, fidaxomicin, rifaximin, amoxicillin/clavulanate, meropenem and piperacillin/tazobactam. Resistance to one or more of tetracycline, moxifloxacin, clindamycin or erythromycin was seen in 26.9% (46 of 171) of isolates, predominantly RTs 126 and 078 of human origin. Fifty per cent of this group were MDR. Human and animal isolates belonging to RTs 033, 237, 281 and 288 presented very similar and generally susceptible phenotypic profiles. Conclusions: Similar pan-susceptible phenotypic profiles seen for a large proportion of human and animal isolates of RTs 033, 237, 281 and 288 support the hypothesis of a common source. Conversely, human strain populations of RTs 126 and 127 show high proportions of antimicrobial resistance and limited phenotypic overlap with their animal counterparts.