We prospectively studied the comparative epidemiology and risk factors for Clostridium difficile, Clostridium perfringens, and Staphylococcus aureus antibiotic-associated diarrhea (AAD). Four thousand six hundred fifty-nine inpatient fecal specimens (11 months) were tested for C. difficile cytotoxin, C. perfringens enterotoxin, and S. aureus by Vero cell assay, enzyme-linked immunosorbent assay, and growth on fresh blood agar, respectively. Two distinct age-, sex-, and location-matched control patient groups were used for multivariate logistic regression risk factor analyses: symptomatic patients who were AAD pathogen negative and asymptomatic patients with histories of recent antimicrobial therapy. All AAD pathogen isolates were DNA fingerprinted. In AAD cases, the prevalences of C. difficile cytotoxin, C. perfringens enterotoxin, and S. aureus were 12.7%, 3.3%, and 0.2%, respectively (15.8% overall). Age of >70 years was a common risk factor. Other risk factors for infective AAD and C. difficile AAD included length of hospital stay and use of feeding tubes (length of stay odds ratios [OR], 1.017 and 1.012; feeding tube OR, 1.864 and 2.808). Female gender and use of antacids were significantly associated with increased risk of C. perfringens AAD (OR, 2.08 and 2.789, respectively), but unlike what was found for C. difficile AAD, specific antibiotic classes were not associated with increased risk. A limited number of genotypes caused the majority of C. difficile and C. perfringens AAD cases. Similar to what was found for C. difficile AAD, there was epidemiological evidence of C. perfringens AAD case clustering and reinfection due to different strains. C. difficile AAD was approximately 4 and 60 times more common than C. perfringens AAD and S. aureus AAD, respectively. Risk factors for these AAD pathogens differed, highlighting the need to define specific control measures. There is evidence of nosocomial transmission in cases of C. perfringens AAD.