Purpose. The purpose of this study was to evaluate the role of scanning electron microscopy (SEM) in the direct diagnosis of suspected onychomycosis with negative mycological test results. Methods. Outpatients diagnosed with suspected onychomycosis with negative mycological test results, including direct microscopic examination with 10% potassium hydroxide (KOH) and fungal culture, on 3 separate occasions were recruited. A small piece of infected nail was obtained for SEM examination. Results. Among the 48 suspected onychomycosis samples, SEM revealed that 18 (37.5%) were positive for fungal structures, including 10 (20.8%) cases of hyphae and 8 (16.7%) cases of yeast blastospores or budding. Conclusion. SEM represents an effective method to diagnose suspected onychomycosis when the traditional mycological methods were negative. Therefore, this technique could be used in clinical practice.