Dermatomycoses are very common worldwide with increasing prevalence. An accurate and rapid detection of fungi is most important for the choice of antimycotics and the success of treatment. The aim of this study was to evaluate a new commercial multiplex-based PCR which allows the detection and differentiation of the most relevant human pathogen fungi causing dermatomycoses in Europe. The accuracy and reproducibility of this application were verified in a clinical performance assessment in comparison to direct microscopy and culture using DNA isolates from 253 clinical samples. Sensitivity, specificity, positive predictive value and negative predictive value of 87.3%, 94.3%, 87.3% and 94.3%, respectively, were calculated for dermatophytes when confirmed by direct microscopy, culture or both. The corresponding values for Candida spp. were 62.7%, 93.5%, 77.8%, and 87.4%, respectively. Furthermore, in comparison to culture, the multiplex PCR was able to detect additional 38 Trichophytum rubrum and 12 Trichophytum interdigitale infections. These results were confirmed by independent PCR analysis. From DNA isolation to diagnosis the multiparameter diagnostic kit gives rise to a 1-day workflow, enables fast clarification of disease aetiology and, thus, contributes to specific therapy selection. The latter is particularly important in light of growing resistance to antimycotics.