The incidence and severity of Clostridium difficile-associated disease (CDAD) is increasing, and standard treatment is not always effective. Therefore, more-effective antimicrobial agents and treatment strategies are needed. We used the agar dilution method to determine the in vitro susceptibility of the following antimicrobials against 110 toxigenic clinical isolates of C. difficile from 1983 to 2004, primarily from the United States: doripenem, meropenem, gatifloxacin, levofloxacin, moxifloxacin, OPT-80, ramoplanin, rifalazil, rifaximin, nitazoxanide, tizoxanide, tigecycline, vancomycin, tinidazole, and metronidazole. Included among the isolates tested were six strains of the toxinotype III, NAP1/BI/027 group implicated in recent U.S., Canadian, and European outbreaks. The most active agents in vitro were rifaximin, rifalazil, tizoxanide, nitazoxanide, and OPT-80 with MICs at which 50% of the isolates are inhibited (MIC(50)) and MIC(90) values of 0.0075 and 0.015 microg/ml, 0.0075 and 0.03 microg/ml, 0.06 and 0.125 microg/ml, 0.06 and 0.125 microg/ml, 0.125 and 0.125 microg/ml, respectively. However, for three isolates the rifalazil and rifaximin MICs were very high (MIC of >256 microg/ml). Ramoplanin, vancomycin, doripenem, and meropenem were also very active in vitro with narrow MIC(50) and MIC(90) ranges. None of the isolates were resistant to metronidazole, the only agent for which there are breakpoints, with tinidazole showing nearly identical results. These in vitro susceptibility results are encouraging and support continued evaluation of selected antimicrobials in clinical trials of treatment for CDAD.