A PCR detecting dermatophytes within a short turnaround time would significantly enhance the management of patients with suspected dermatophytosis. This study aimed at comparing the results of a real-time PCR assay with those of the conventional diagnostic (direct microscopy and culture) performed by a dermatologist working in a medical mycology laboratory for the detection of dermatophytes in nail and skin samples.A total of 112 specimens (54 nail and 58 skin) were collected from 52 patients with one to four suspected dermatophytosis lesions. The PCR diagnostic indices were calculated for either sample- or patient-based dermatophytosis diagnosis. The sample-based diagnostic efficacy yielded 79% sensitivity and 73% specificity. The patient-based diagnostic efficacy was higher with 100% sensitivity and 82% specificity. Interestingly, PCR yielded significantly (p<0.004) lesser false negative results and performed overall better (diagnostic odds ratio=24.0 vs. 5.5) in nail than in skin samples. In conclusion, this real-time PCR assay performance was consistent with those of the conventional methods in the hands of a skilled expert and particularly efficacious in diagnosing dermatophyte onychomycosis. This PCR is suited to high throughput batch processing; if used instead of direct microscopy, it could reduce hands-on time in the routine clinical laboratory workflow.