The antimicrobial susceptibility and virulence factors of Clostridium difficile clinical isolates in Taiwan have not previously been reported. One hundred and thirteen isolates were collected from two major teaching hospitals in Taiwan from 2001 to 2009. Molecular typing was performed by an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA) and PCR ribotyping. Detection of tcdA, tcdB, cdtA, and cdtB genes was performed using a multiplex PCR assay, and gyrA and gyrB genes of moxifloxacin-nonsusceptible isolates were sequenced. All isolates were susceptible to vancomycin and metronidazole. Ninety-five (84%) isolates were susceptible to moxifloxacin, and the MIC(90) for nemonoxacin was 4 μg/ml. Tigecycline showed favorable antibacterial activity (MIC(90) of 0.06 μg/ml). Thirteen rep-PCR types were identified as a predominant rep-PCR type (type A; non-North American pulsed-field gel electrophoresis type 1 [NAP1], -NAP7, or -NAP8) accounting for 52.2% (59 isolates). Nine of 18 moxifloxacin-nonsusceptible isolates belonged to the rep-PCR type A. The rep-PCR type A and C isolates were distinct from NAP1 (ribotype 027) and NAP8 (ribotype 078) as determined by PCR ribotyping. Seventy-four (65%) isolates harbored tcdA and tcdB, and 15 (13%) harbored cdtAB encoding binary toxin. Eleven isolates had a gene deletion in tcdC, including a 39-bp deletion (9 isolates) and an 18-bp deletion (2). In conclusion, dissemination of a predominant C. difficile clone in southern and northern Taiwan was noted. However, no NAP1 (ribotype 027) isolate could be discovered in this study.