Clostridioides difficile is the most common anaerobic bacterium that causes healthcare-associated infections, and prompt diagnosis and infection control are important because it causes C. difficile infection (CDI). In this evaluation, the C. difficile nucleic acid detection reagent, Smart Gene CD Toxin B (Mizuho Medy Co., Ltd., hereinafter referred to as the "evaluation reagent") was evaluated for its clinical performance in comparison with real-time PCR and toxigenic culture (TC). Measurement of evaluation reagents and real-time PCR were performed on 157 residual stool specimens from suspected CDI patients. For TC, stool culture was performed, and colonies in which C. difficile was identified by a mass spectrometer (MALDI Biotyper) were checked for toxin production using a rapid antigen diagnostic kit. The results of the evaluation reagents showed a high concordance rate; 100% sensitivity (81/81) and 100% specificity (76/76) with real-time PCR, 89.8% sensitivity (79/88), and 97.1% specificity (67/69) with TC. The evaluation reagent enables a simple nucleic acid amplification test (NAAT) in a short time and is thought to be useful in CDI treatment, which requires rapid diagnosis and infection control.