The anthropophilic dermatophyte Trichophyton tonsurans and its zoophilic counterpart T. equinum are phylogenetically closely related. The barcoding marker rDNA internal transcribed spacer (ITS) shows limited variation between these two species. In the current study, we combined molecular approaches with phenotypic data to determine the species boundaries between T. tonsurans (n = 52) and T. equinum (n = 15) strains originating from humans (n = 40), horses (n = 26), and a mouse (n = 1). Culture characteristics and physiology on Trichophyton agar media 1 and 5 were evaluated. Multi-locus sequencing involving ITS, partial large rDNA subunit (LSU), β-tubulin (TUB), 60S ribosomal protein (RPB), and translation elongation factor-3 (TEF3) genes, and the mating-type (MAT) locus was performed. Amplified fragment length polymorphism data were added. None of the test results showed complete mutual correspondence. With the exception of strains from New Zealand, strains of equine origin required niacin for growth, whereas most strains from human origin did not show this dependence. It is concluded that T. tonsurans and T. equinum incompletely diverged from a common lineage relatively recently. MAT1-1 and MAT1-2 are the main distinguishing genes between the two species.