Background: One of the most prevalent nail conditions is onychomycosis, that is mostly brought on by dermatophyte fungi, non-dermatophyte molds (NDMs) and yeast which may be confused with other nail lesions so, it is crucial to accurately identify the causative agent to initiate proper medications. Fungal culture and K OH microscopy have always been the gold standards for diagnosis. Aim: This study aimed to compare the results of a multiplex real-time PCR (RT-PCR) assay with those of conventional diagnostic methods (direct microscopy and culture) in detecting and diagnosing onychomycosis. Methods: A total of 100 nail samples from clinically suspected onychomycosis patients were divided into 3 pieces, one nail piece was set aside for microscopy, another for culture and the rest were kept at room temperature in sterile screwed vials in preparation for the multiplex RT-PCR test and DNA extraction. Results: of 100 nail samples, 30 had negative direct K OH and 70 had positive direct K OH. In the group of negative K OH samples, 10/30 had a positive culture. In the group of positive K OH samples, 70/100 had a positive culture. Multiplex RT-PCR demonstrated 92.8% sensitivity, 62.7% specificity, 61.9% VPP and 93.1% VPN. Conclusion: using multiplex RT-PCR can improve the detection of onychomycosis and decrease the turn round time which could improve the disease outcome.