Purpose <p>Dermatomycoses represent the most frequent form of human fungal infection globally. The conventional diagnostic approach to identify dermatomycoses is based on direct microscopic examination (DME) and fungal culture. However, these procedures suffer from a number of significant limitations. We aim to evaluate the clinical performance of the novel Dermato<Emphasis Type="BoldItalic">Plex</Emphasis> qPCR assay (SpeeDx) in comparison to conventional methods for the detection of dermatophyte fungi and <i>Candida</i> spp. in skin and nail material.</p> Methods <p>Between February and July 2024, 1320 samples from 1252 patients were collected for the investigation of dermatophyte fungi at the Navarra University Hospital in Spain. DME, culture and DNA extraction were systematically executed in all specimens, and the qPCR assay was retrospectively tested. The Novaplex™ Dermatophyte qPCR assay (Seegene) was used in case of discrepancies.</p> Results <p>The Dermato<Emphasis Type="BoldItalic">Plex</Emphasis> qPCR assay performed well for the molecular detection of dermatophyte fungi and <i>Candida</i> spp. in skin and nail material; with an overall agreement of 75.4% when compared with culture. Among discrepancies, the majority (75.6%) were ultimately confirmed as true-positives, while only 12.4% resulted in false-negative results. Overall, sensitivity of the qPCR method for the diagnosis of dermatomycoses was 2.3x and 1.7x greater (1.7x and 1.6x for dermatophytosis) when compared with DME and culture, respectively.</p> Conclusion <p>This is the largest clinical evaluation of a qPCR method for the diagnosis of dermatomycoses. Considering the greater sensitivity of molecular techniques over conventional procedures, the Dermato<Emphasis Type="BoldItalic">Plex</Emphasis> assay emerges as a reliable and promising qPCR method for the diagnosis of cutaneous mycoses.</p>