Clostridium difficile is the most common agent of nosocomial bacterial diarrhoea in adults. In 2006, C. difficile outbreaks were described in France with the highly virulent strain PCR-ribotype 027, which is also resistant to moxifloxacin and erythromycin. The aim of this study is to perform a phenotypic and molecular characterization of C. difficile strains isolated in Jean-Verdier-René-Muret hospitals. Thirty three C. difficile toxigenic strains isolated in symptomatic patients from 2001 to 2007 were studied. Toxins A and B detection was performed with an immunoenzymatic method (ICTAB, Meridian). The agar diffusion method was performed for determination of antibiotic susceptibility for metronidazole, vancomycin, erythromycin and moxifloxacin. The E-test was performed for determination of metronidazole, vancomycin and tigecycline MIC. Binary toxin genes cdtA and cdtB were detected by PCR. PCR-ribotyping was performed according to Bidet et al. From 2001 to 2007, all the isolates studied were susceptible to metronidazole, vancomycin and tigecyclin. We observed a significant decrease of susceptibility to moxifloxacin (100% in 2001 versus 28.5% in 2007) and to erythromycin (60% in 2001 versus 14% in 2007). Toxins A/B were detected in all the isolates. Fifteen per cent of the isolates studied produced the binary toxin not correlated with a specific PCR-ribotype. Ribotype 18 was the most prevalent PCR-ribotype detected since 2006. The isolates displaying this PCR-ribotype were resistant to erythromycin and moxifloxacin and were principally isolated in the same ward, suggesting cross infection. This study showed that: (1) over a six-year period, the susceptibility to metronidazole and vancomycin remained stable; (2) different clones of C. difficile circulated during these six years. Recently an epidemic strain resistant to erythromycin and moxifloxacin of ribotype 18 has emerged in the gastroenterology unit where fluoroquinolones are frequently used demonstrating the role of antibiotic selection pressure. The emergence of these isolates could explain the significant decrease of susceptibility to moxifloxacin and erythromycin observed in 2007. However, today, no isolate with a PCR-ribotype 027 was detected.