The prevalence, incidence, and epidemiologic factors of Clostridium difficile-associated diarrhea (CDAD) in a Peruvian hospital were studied. We conducted a cross-sectional study in patients > 14 years of age from medical/surgical wards of the Cayetano Heredia National Hospital (Lima, Peru) from September 2005 to May 2006. CDAD was defined in a case of nosocomial diarrhea when C. difficile toxin A and/or toxin B was detected by enzyme immune assay (EIA) in stools. A total of 4,264 patients were admitted, with 156 (3.7%) developing nosocomial diarrhea. Fifty-five of 156 (35.2%) cases of nosocomial diarrhea were diagnosed as CDAD. The overall incidence per 1,000 admissions was 12.9. Multivariate analysis showed that use of diapers (OR, 3.54; 95% CI, 1.71-7.34; P = 0.001) and presence of another patient with CDAD housed in the same room (OR, 2.97; 95% CI, 1.14-7.76; P = 0.026) were significantly associated with CDAD. Hospital transmission of C. difficile commonly occurred, supporting infection-appropriate measures directed toward the reduction of CDAD in low-resource settings. Copyright © 2007 by The American Society of Tropical Medicine and Hygiene.