Background: Clostridium spp. spores are resistant to many factors, including alcohol-based disinfectants. The presence of clostridial spores in a hospital environment may lead to infection outbreaks among patients and health care workers. Objective: This study aimed to detect clostridial spores in aurology hospital using C diff Banana Broth™ and assess the antibiotic sensitivity and toxinotypes of isolates. Methods: After diagnosing COVID-19 in medical staff and closing an 86-bed urology hospital in 2020 for H2O2 fogging, 58 swabs from the hospital environment were inoculated to C diff Banana Broth™, incubated at 37°C for 14 days, checked daily, and positive broths were sub-cultured anaerobically for 48 h at 37°C. After identification, multiplex PCR (mPCR) was performed for Clostridium perfringens, C. difficile toxin genes, and MIC determination. Results: In this study, 16.58 (~ 28%) strains of Clostridium spp. were cultured: 11 - C. perfringens, 2 - C. baratii, and 1 each of C. paraputrificum, C. difficile, and C. clostridioforme. Moreover, C. difficile produced all toxins, and 11 C. perfringens consisted of 1 cpa, 7 cpb2, 2 cpiA, and 1 cpb gene-positive. All isolates were sensitive to metronidazole, vancomycin, moxifloxacin, penicillin/tazobactam, and rifampicin. Two out of the 11 C. perfringens strains were resistant to erythromycin and clindamycin. Conclusions: Regardless of the performed H2O2 fogging, antibiotic-resistant, toxigenic strains of C. perfringens (69%) obtained from the urology hospital environment were cultured using C diff Banana Broth™, indicating the need to develop the necessary sanitary and epidemiological procedures in this hospital.