We recently reported a high prevalence of Clostridium difficile in retail vegetables, compost and lawn in Western Australia. The objective of this study was to investigate the antimicrobial susceptibility of previously isolated food and environmental C. difficile isolates from Western Australia. A total of 274 C. difficile isolates from vegetables, compost and lawn were tested for susceptibility to a panel of 10 antimicrobial agents (fidaxomicin, vancomycin, metronidazole, rifaximin, clindamycin, erythromycin, amoxicillin/clavulanic acid, moxifloxacin, meropenem and tetracycline) using the agar incorporation method. Fidaxomicin was the most potent agent (MIC50/MIC90, 0.06/0.12 mg/L). Resistance to fidaxomicin and metronidazole was not detected and resistance to vancomycin (0.7%) and moxifloxacin (0.7%) was low. However, 103 isolates (37.6%) showed resistance to at least one agent, and multidrug resistance was observed in 3.9% of the resistant isolates (4/103), all of which came from compost. A significantly greater proportion of compost isolates were resistant to clindamycin, erythromycin and tetracycline compared with food and/or lawn isolates. Clostridium difficile ribotype (RT) 014/020 showed greater clindamycin resistance than other less common RTs (P = 0.008, χ2). Contaminated vegetables, compost and lawn could be playing an intermediary role in the transmission of C. difficile from animals to humans. Environmental strains of C. difficile could also function as a reservoir for antimicrobial resistance genes of clinical relevance. This study provides a baseline for future surveillance of antimicrobial resistance in environmental C. difficile isolates in Australia.