Dermatophytosis is a superficial fungal infection of keratinized tissues. Dermatophytes can cause discomfort but are not usually life threatening. However, the infection can spread and may lead to systemic fungal infections in immunocompromised patients. Currently available diagnostic methods include potassium hydroxide (KOH) testing and periodic acid–Schiff (PAS) staining. However, most diagnostic techniques cannot be performed rapidly; days to weeks may be required for conclusive results. Certain dermatophytes autofluoresce and can be observed under fluorescence microscopy. The authors examined a series of 24 cases of hematoxylin and eosin–stained dermatophytoses using fluorescence microscopy and compared the results with those obtained using PAS staining. The diagnostic performance of fluorescence microscopy was better than that of PAS staining. Fluorescence microscopy allowed the detection of all the cases that were detected using PAS staining. In addition, fluorescence microscopy facilitated the detection of weak fluorescence in 2 cases with ambiguous PAS results. These results support the integration into clinical practice of fluorescence microscopy as a simple and rapid diagnostic tool for evaluating cases of suspected dermatophytosis.