Objective: To determine the prevalence and antimicrobial resistance of Clostridium difficile (C. difficile) isolated from ready-to-eat foods of Iran. Methods: From January to August 2013, a total of 368 unpacked ready-to-eat food samples were purchased from randomly selected supermarkets, retail stores and restaurants located in Isfahan and Shahrekord, Iran and were evaluated for the presence of C. difficile. Results: C. difficile spores were detected in 5 (1.36%) of the 368 samples. The highest prevalence of C. difficile was found in fasl salad (4.29%), followed by yogurt stew (2%), and olovyeh salad (0.93%). All 140 maccaroni salad and falafel sandwich samples were negative for C. difficile. One of the five C. difficile isolates (20%) contained tcdA, tcdB and cdtB toxin genes and four strains (80%) contained tcdA, and tcdB toxin genes. Also, among the five C. difficile isolates, only three strains were found to be toxigenic for toxin A and/or B by ELISA. Isolates were susceptible to vancomycin and metronidazole, but variably resistant to other antimicrobial drugs. Conclusions: This study, combined with studies on other food sources, suggests that widespread contamination of food is common.