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The objective of this study was to evaluate antimicrobial susceptibility in Clostridium difficile strains isolated from animals and humans in Brazil. The 54 C. difficile strains used were isolated from stool samples from piglets (n=16), dogs (n=13), humans (n=13), foals (n=8) calves (n=2), an ocelot (n=1) and a maned wolf (n=1). Antimicrobial susceptibility was determined using the serial plate agar dilution method for penicillin, florfenicol, oxytetracycline, erythromycin, vancomycin, metronidazole and tylosin. The C. difficile strains assessed were susceptible to metronidazole and vancomycin. Florfenicol resistance was rarely observed; 52 (96.4%) strains were sensitive to this antimicrobial. Five (9.3%), five (9.3%), 14 (25.9%) and 20 (37.0%) strains were resistant to oxytetracycline, penicillin, tylosin and erythromycin respectively.