Background: Clostridium difficile infection (CDI) is a significant cause of morbidity and mortality in both the acute care setting and the wider healthcare system. The purpose of this study was to evaluate the in vitro activity of fidaxomicin against C difficile isolates from a university teaching hospital in China. Methods: One hundred and one C difficile isolates were collected and analyzed for toxin genes by multiplex PCR. The toxin gene positive strains were also typed by multilocus sequence typing (MLST) and PCR-ribotyping. The MICs of the isolates were determined against fidaxomicin, metronidazole, vancomycin, tigecycline and moxifloxacin, by the agar dilution method. Results: All the 101 isolates exhibited low MICs to fidaxomicin (0.032-1 mg/L), metronidazole (0.125-1 mg/L), vancomycin (0.25-2 mg/L) and tigecycline (0.016-0.5 mg/L). Tigecycline showed the lowest geometric mean MIC value (0.041 mg/L), followed by fidaxomicin (0.227 mg/L), metronidazole (0.345 mg/L), and vancomycin (0.579 mg/L). About 35% of the strains (n = 35) were resistant to moxifloxacin, and the resistance rate to moxifloxacin for A-B+CDT- isolates (85.0%) was much higher than that of A+B+CDT- (15.7%) and A-B-CDT- (29.2%) isolates (P < 0.001). The MIC values of fidaxomicin, metronidazole, vancomycin and moxifloxacin against the 3 ST1 isolates were higher than for other STs. All the 28 moxifloxacin-resistant toxigenic isolates carried a mutation either in gyrA or/and gyrB. Conclusion: Fidaxomicin exhibited high antimicrobial activity against all C. difficile isolates tested, which shows promise as a new drug for treating Chinese CDI patients.