Dermatophytes and other members of Onygenales are unique in their ability to degrade keratin, affecting hair and nails, and in case of human hosts cause infection of the skin. Subtillisins are essential proteases in keratin assimilation, and subtilisin-like protease 1 (SUB1) and SUB3–7 are specific for dermatophytes. eIF2α kinases are serine-threonine kinases that perform essential functions in response to infection, proteotoxicity, and nutrient scavenging. The relatively conserved nature of EIF2AK4 among fungi makes them potential evolutionary markers, which may contribute to a deeper understanding of dermatophyte taxonomy and evolution. The aim of the present study was to evaluate the phylogeny of dermatophytes using EIF2AK4 and SUB1 genes, compared to ITS evolution. The EIF2AK4 tree had a similar topology to the SUB1 tree, and both deviated from the ITS gold standard by evolution. Our preliminary findings with a limited dataset suggest that the EIF2AK4 and SUB1 genes provide a reasonably correct reflection of the evolution of Arthrodermataceae. In addition, the study analyzed in vitro keratinolytic responses of 19 dermatophyte species using hairs of a broad selection of mammals as substrates. Trichophyton mentagrophytes and Nannizzia gypsea were the most active in degrading hair, while Trichophyton verrucosum, Trichophyton tonsurans and Epidermophyton floccosum showed low response. Hairs of Hyracoidea and Rodentia were most affected of all mammal hairs, while in contrast, bat hairs were difficult to degrade by nearly all tested dermatophyte species. Zoophilic species showed more activity than anthropophilic dermatophytes, but hair degradation profiles were not diagnostic for particular dermatophyte species.