A total of 65 toxigenic Clostridium difficile strains isolated from patients with antibiotic-associated diarrhea were tested for susceptibility to avilamycin, flavomycin, monensin, and salinomycin. Except for flavomycin the substances showed in vitro efficacy comparable to reports of the currently most commonly used drugs for treatment of C. difficile. This indicates that these old compounds may be useful for the treatment of C. difficile infections in man and perhaps for other bacterial causes of diarrhea.