Clostridium difficile remains the leading cause of nosocomial-acquired diarrhea. This study investigated antimicrobial susceptibility patterns of C. difficile over a 3-year period. Three hundred seventeen C. difficile isolates recovered between 2002 and 2004 were analyzed for their susceptibility to erythromycin (ERY), clindamycin (CLI), moxifloxacin (MXF), doxycycline (DOX), vancomycin (VAN), and metronidazole (MTR) by Etest. The molecular basis for resistance was investigated using polymerase chain reaction (PCR) and DNA sequencing. PCR ribotyping was used to differentiate strains. All strains were susceptible to VAN and MTR. Resistance rates to ERY/CLI, MXF, and DOX increased during the study period. Eighty-four (26.5%) strains exhibited resistance against ERY/CLI, MXF, and DOX. Prevalence of resistance genes was as follows: ermB, 83; ermQ, 0; ermFS, 1; tetM, 84; tetP, 0; tetO, 2; and gyrA mutation, 76. These results indicate an increasing trend in the prevalence of combined resistance against macrolide-lincosamide-streptogramin B antibiotics, fluoroquinolones, and tetracycline in C. difficile. The lack of understanding of antibiotic resistance mechanisms in C. difficile and the increased resistant strains warrants further investigations.