Clostridium difficile (recently Clostridioides difficile) is a leading cause of hospital- and antimicrobial-associated diarrhea (AAD). The present study was carried out to investigate the prevalence of toxigenic C. difficile, antibiotic resistance and its associated risk factors in Iranian hospitalized patients. This cross-sectional study was conducted from October 2017 to June 2018 in three teaching hospitals in southwestern Iran. During this period, a total of 215 non duplicated nosocomial AAD samples were collected from the hospitalized patients older than two years of age. Presumptive C. difficile isolates were identified by standard microbiologic methods and confirmed by specific PCR primers. The minimum inhibitory concentrations (MICs) were determined by the agar dilution method. PCR was carried out to determine the presence of toxin genes (tcdA, and tcdB). In all, from the 215 diarrheal samples, the frequency of C. difficile culture-positive samples was 21.4% (n = 46). Of the 46 C. difficile isolates, 43 carried both toxins, two isolates only had the tcdB gene, and one was negative for both toxins. Overall, all isolates of C. difficile were susceptible to metronidazole and vancomycin. The MIC50/MIC90 of metronidazole and vancomycin were 0.75/2 μg/mL, 0.25/0.75 μg/mL, respectively. The findings of this study show the prevalence of CDI in hospitalized patients in southwestern Iran, highlighting the importance of active surveillance of CDI in hospitals. Meanwhile, all of the tested isolates were susceptible to metronidazole and vancomycin, which encourages the use of these antibiotics as the drug of choice for initial treatment of CDI in our region.