Abstract：Objective To investigate the antibacterial activity in vitro of metronidazole combined with three antibiotics（linezolid，fidaxomycin and vancomycin）in clinical isolates of Clostridium difficile，and to provide experimental basis for combination therapy. Methods The metronidazole and other three kinds of anti-inflammatory drugs in vitro and in combination were applied to 32 Clostridium difficile strains，by agar double dilution method，MIC was calculated to calculate the fractional antibacterial concentration（FIC）index，draw bactericidal curve. Anti-inflammatory activity in vitro of metronidazole and other three kinds of antibacterial drugs were evaluated. Results Linezolid，fidagamicin，vancomycin combined with metronidazole respectively in vitro，MIC values were lower with the combination of linezolid and fidaxomycin than alone，the combined susceptibility index were all less than 2；Clostridium difficile killed speed with combined application was faster，the effect increased. MIC value of vancomycin combined with metronidazole was no significant changed. Conclusion Linezolid and fidaxomycin combined with metronidazole in vitro had a synergistic antibacterial effect，which could reduce the MIC against Clostridium difficile，but MIC was no significant change in the vancomycin combined with metronidazole.