Toxin detection from stool specimens is critical for the diagnosis of Clostridium difficile-associated diarrhea (CDAD). In Japan, only two toxin detection kits targeting toxin A alone are commercially available. We evaluated ImmunoCard Toxin A & B (ImmunoCard), based on enzyme immunoassay for the rapid detection of both C. difficile toxins A and B in stool specimens, compared to a toxin A detection kit (Uniquick) and cytotoxin assay. C. difficile was also cultured from stool specimens and the toxin production type of C. difficile isolates was identified by PCR analysis. Compared to cytotoxin assay, ImmunoCard sensitivity was 86.2%, specificity 93.8%, positive predictive value 91.8%, and negative predictive value 89.4% (n = 146). Sensitivity was significantly higher than that of Uniquick (60.0%, p = 0.0016). ImmunoCard detected 90.6% of cytotoxin positive specimens with isolated toxin A-positive, toxin B-positive C. difficile strains (Uniquick; 67.9%, p = 0.008) and 70.0% of these with isolated toxin A-negative, toxin B-positive C. difficile strains. Although ImmunoCard was slightly less sensitive than cytotoxin assay, it requires no special equipment and completes the entire test in up to 20 min. ImmunoCard thus appears very useful in the rapid diagnosis of CDAD in the clinical laboratory. Kits for the detection of both C. difficile toxins A and B should be immediately introduced into Japan to ensure the correct diagnosis of CDAD and infection control.