Onychomycosis is mainly caused by two dermatophyte species, Trichophyton rubrum and Trichophyton interdigitale. A study of nail invasion mechanisms revealed that the secreted subtilisin Sub6, which has never been detected under in vitro growth conditions, was the main protease secreted by T. rubrum and T. interdigitale during infection. In contrast, most of the proteases secreted during the digestion of keratin in vitro were not detected in infected nails. The hypothesis that proteases isolated from dermatophytes grown in a keratin medium are virulence factors is no longer supported. Non-dermatophyte fungi can also be infectious agents in nails. It is necessary to identify the infectious fungus in onychomycosis to prescribe adequate treatment, as moulds such as Fusarium spp. and Aspergillus spp. are insensitive to standard treatments with terbinafine or itraconazole, which are usually applied for dermatophytes. In these refractory cases, topical amphotericin B treatment has shown to be effective. Terbinafine treatment failure against dermatophytes is also possible, and is usually due to resistance caused by a missense mutation in the squalene epoxidase enzyme targeted by the drug. Trichophyton resistance to terbinafine treatment is an emerging problem, and a switch to azole-based treatment may be necessary to cure such cases of onychomycosis.