Background: Clostridium difficile is the primary reason of the nosocomial diarrhea. The antimicrobial therapy plays a central role in the pathogenesis of Clostridium difficile associated diarrhea (CDAD). Although nearly all classes of antimicrobial agents have been associated with CDAD, clindamycin and the third-generation cephalosporins have traditionally been considered to the greatest risk factor. Recent studies have also implicated fluoroquinolones as high-risk agents due to increasing use of the agents. This study was to determine the incidence and the risk factors of CDAD related to the administered antibiotics and to assess the therapeutic regimen of metronidazole or vancomycin based on the C. difficile toxin assay Methods: A retrospective study was performed in patients with Clostridium difficile toxin assay at I Hospital (Incheon, South Korea) during the period from January 2007 through December 2007. Administrative, laboratory, and pharmacy data were collected from Electronic Medical Databases. Results: The analysis included 129 reported C.difficile toxin assay results, with 42 positive cases and 87 negative cases. Significant antibiotic risk factors for CDAD included the use of the fourth-generation cephalosporinse (OR=5.97, 95% CI 1.37-25.98, P=0.017). Administration of metronidazole was protective against CDAD (OR=0.30, 95% CI 0.12-0.74, P=0.009). Prolonged antimicrobial therapy has been associated with an increased risk of CDAD. The third-generation cephalosporins (OR=3.81, 95% CI 1.08-13.41, P=0.037) and aminoglycoside (OR=5.50, 95% CI 1.43-21.10, P=0.013) demonstrated greater risk for CDAD over 15 days than 8days or less days of treatment duration. Conclusions: The fourth and third generation cephalosporin, aminglycoside were the significant risk factors compared with other antibiotics, whereas metronidazole appears to be protective. The longer duration of antiobiotic use increased CDAD.