BACKGROUND: Onychomycosis is worldwide the most prevalent infection of the nail. It is mainly caused by the dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes and to a lesser extent Trichophyton tonsurans. The yeast Candida albicans and the mould Scopulariopsis brevicaulis can also cause onychomycosis. Management of these nail conditions may require appropriate treatment methods and therefore the identification of the causative species can be of importance. However, the determination of agents causing onychomycosis is still not optimal. OBJECTIVES: To detect and differentiate causative organisms of onychomycosis in an ex vivo nail model by means of Raman spectroscopy. The work focusses is on the discriminative power of Raman spectroscopy for detection of differences between T. rubrum, T. mentagrophytus and T. tonsurans on human nail and distinguishing these dermatophytic from the non-dermatophytic species S. brevicaulis and C. albicans. METHODS: Raman spectra (200/sample) were taken from 50-µm human nail slices infected with T. rubrum, T. mentagrophytus, T. tonsurans, S. brevicaulis or C. albicans using a 2500 High-Performance Raman Module and 785-nm diode laser. Processed spectra were analysed by sorting the correlation matrix and presented as dendrogram and heat map. Raman spectra from suspended dermatophytic microconidia were taken for mutual comparisons. RESULTS: Spectral differences between the dermatophytes T. rubrum, T. mentagrophytus and T. tonsurans (635-795, 840-894, 1018-1112, 1206-1372, 1566-1700/cm) and the non-dermatophytes S. brevicaulis and C. albicans (442-610, 692-758, 866-914, 1020-1100, 1138-1380,1492-1602/cm) growing on nail were confirmed by clustering correlation showing two main clusters. Dissimilarities between tested dermatophytes were also found with T. rubrum being most different. Raman spectra of the dermatophytic microconidia varied over the whole tested 400-1800/cm range. CONCLUSION: Important dermatophytic and non-dermatophytic agents of onychomycosis growing on ex vivo human nail can be distinguished specifically and non-invasively by Raman spectroscopy.