Background: Onychomycosis is a common nail disorder caused mostly by Trichophyton rubrum and Trichophyton mentagrophytes. As other conditions may resemble onychomycosis, the accurate detection and identification of the causal agent is mandatory. Conventional methods for detecting fungi in nail specimens are either nonspecific or insensitive and time consuming, especially in cases of previous antifungal therapy. Recently, PCR based assays have been introduced to improve onychomycosis diagnosis. Objectives: Evaluation of the performance of a multiplex PCR for detecting demartophytes compared with the results of the conventional culture technique in patients with clinically suspected onychomycosis. Methodology: A multiplex PCR-based method was evaluated using 50 nail specimens collected from onychomycosis patients. After a rapid DNA extraction method, the multiplex PCR was performed using three sets of primers aimed at detecting dermatophytes generally, T. rubrum and T mentagrophytes. Amplicon analysis was made using agarose gel electrophoresis. PCR results were compared with those of the direct microscopy and culture of the nails. Results: Among the 50 patients with onycomycosis, 70% (35/50) were positive for fungal elements microscopic examination. Positive fungal cultures were detected in 60% (30/50). The results of PCR showed that 58% (29/50) were positive by pan Derm PCR. Dermatophyte positive by PCR were 23 T. rubrum, 4 T. mentagrophytes and 2 other dermatophytes, while 20/50 (40%) of them yielded growth of dermatophytes in culture, 17 T. rubrum and 3 T. mentagrophytes. In ten samples where culture yielded growth of non-dermatophyte moulds and yeasts, PCR was positive for T. rubrum (one) while PCR was negative in 9 non-dermatophyte mould and yeast positive cultures. Conclusion: Multiplex-PCR method applied directly on nail specimens could be a promising diagnostic tool for the management of the patients with suspected onychomycosis.