Clostridium perfringens has emerged as an important cause of antibiotic-associated diarrhea (AAD), particularly in the hospital environment. Here we investigated the prevalence and molecular epidemiology of C. perfringens isolated from 2280 fecal samples from Iranian diarrheal patients suspected of having AAD. Overall, AAD was diagnosed in 13.3% (303/2280) of patients and associated with advanced age (>50 years, P = 0.001). A total of 106 C. perfringens isolates were cultured from AAD (n = 68) and non-AAD (n = 38) groups, with toxinotypes A and F comprising 84% and 16% of isolates, respectively. Notably, 41.2% of type F strains were also cpb2-positive and enterotoxigenic cpe-positive strains were detected in 13.2% of the isolates from AAD patients. Genes associated with the VirR/VirS signal transduction (virR, virS) and accessory gene regulator (agrB, agrD) systems were detected in 56.6% and 67% of the isolates, respectively, and peptides of the quorum-sensing modulator, AgrD were highly conserved across all strains. The high prevalence of C. perfringens in Iranian AAD patients suggests that diagnostic laboratories in this region should consider screening for C. perfringens in cases of suspected AAD, especially if the specimen is negative for other pathogens and/or the patients are aged >50 years.