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Background: Onychomycosis is a common nail disease that accounts for 50% of abnormal nails. Accurate diagnosis of onychomycosis is essential before long-term antifungal treatment. Reflectance confocal microscopy (RCM) is a rapid real-time imaging technique that is widely used for the diagnosis of skin diseases. Objective: The purpose of this study was to compare the effectiveness of RCM and standard diagnostic methods in the diagnosis of onychomycosis and to determine their sensitivity and specificity. Methods: We evaluated 60 Korean patients with suspected onychomycosis using three diagnostic methods: KOH smear, culture, and RCM. RCM diagnosis was based on the presence of specific structures and filamentous and/or spore-like spherical structures. To compare the usefulness of the diagnostic methods, the sensitivity, specificity, positive predictive value, and negative predictive value for each method were calculated. Results: Of the patients, 57 had at least one positive value for these diagnostic methods. RCM was the most sensitive test for the diagnosis of onychomycosis, with a sensitivity of 84.21%, specificity of 100%, positive predictive value of 100%, negative predictive value of 25%, and accuracy of 85%. Among the 48 patients who showed positive results on RCM, RCM images presented hyphae structures in 35 cases (72.9%), conidia structures in 39 cases (81.3%), and coexistence of both structures in 26 cases (54.2%). Conclusion: In this study, RCM was the most sensitive method for the diagnosis of onychomycosis. The fungal morphology and load can be identified in vivo by RCM and can be used in clinical situations.