One hundred thirty-three Clostridium difficile strains were collected from 71 patients and analyzed for the presence of C. difficile toxin B by the cell cytotoxicity neutralization assay, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped and analyzed for sporulation frequency. The MICs of the isolates were determined against cadazolid and seven other antimicrobial agents by the agar dilution method. All isolates were positive for toxin B by the cell cytotoxicity neutralization assay. One hundred fourteen isolates were positive for toxin A and B and 16 isolates were positive for toxin A, toxin B and binary toxin by PCR. Three isolates were negative for toxin A but positive for toxin B. Thirty-three different ribotypes were identified. No strain of ribotype 027 was found. No differences in sporulation were noticed between the primary and recurrent isolates. All 133 isolates were sensitive to cadazolid (0.064–0.5 mg/l), fidaxomicin (0.008–0.125 mg/l), metronidazole (0.125–2 mg/l), vancomycin (0.125–1 mg/l) and tigecycline (0.032–0.25 mg/l). Three isolates were resistant to linezolid (8 mg/l), 15 isolates were resistant to moxifloxacin (8–32 mg/l) and 103 isolates were resistant to clindamycin (8–256 mg/l). No association between toxins A, B and binary toxin, ribotypes or the sporulation and the sensitivity to cadazolid could be found. Cadazolid has a potent in vitro activity against C. difficile.