Clostridium difficile infection (CDI) is changing as evidenced by increasing virulence, rising incidence, unresponsiveness to metronidazole therapy, and worse outcomes. Thus, it is critical that CDI diagnosis be accurate so ongoing epidemiology, disease prevention, and treatment remain satisfactory. We tested 10 diagnostic assays, including 1 commercial real-time polymerase chain reaction (qPCR) test for the laboratory detection of toxigenic C difficile on 1,000 stool samples. Sensitive culture for toxigenic C difficile using 2 types of media with broth enrichment defined the reference standard. For the study, 1,000 tests were performed on samples from 919 patients. Of the samples, 146 contained evidence for toxigenic C difficile and represented the true-positive results. Only the US Food and Drug Administration-cleared qPCR assay (Becton Dickinson, Franklin Lakes, NJ) and 1 glutamate dehydrogenase test (TechLab, Blacksburg, VA) were not statistically inferior to culture in sensitivity. The common enzyme immunoassay tests all had sensitivity values less than 50%. Clinical laboratory professionals need to seriously consider their diagnostic testing and use the assays that perform best for the detection of CDI.