Background: Onychomycosis is a critical medical problem. This fungal infection of the nails leads to disfigurement, pain and impaired quality of life. Given that it requires long-term expensive therapy; a proper diagnosis of this infection is greatly demanded. Aim: To address onychomycosis regarding clinical and laboratory findings, and investigate different laboratory methods used in the identification of dermatophytes implicated in onychomycosis; including conventional laboratory methods (KOH preparation), culture and multiplex polymerase chain reaction (PCR). Methods: This cross-section study included sixty-one (61) patients (82% females and 18% males). All collected samples were investigated using microscopic examination and cultivation of samples. In addition, DNA extraction and PCR amplification were evaluated. Results: After mycological study of 61 cases, we found that distal lateral subungual onychomycosis (DLSO) was the most common clinical type in our study. Also, the dermatophytes appeared to be the chief causative agent in onychomycosis (61%). In addition, positive results were identified in 67.2%, 60.7% and 73.8% of the studied group using PCR, culture and, KOH direct microscopy, respectively. With respects to the results of PCR in the study, the sensitivity, specificity and accuracy were, 100%, 83.3% and 93.4% respectively. Conclusion: PCR was a highly sensitive method for diagnosis of onychomycosis. The application of PCR technology directly to the clinical specimens will permit early and accurate diagnosis of onychomycosis, and leading to prompt initiation of specific antifungal therapy