Onychomycosis is a common and intractable superficial mycosis that occurs worldwide. Treatment with both oral and topical drugs is recommended, but the objective evaluation procedure to determine the efficacy of and the appropriate delivery system for the drugs remains controversial. This may be attributed to the lack of a reliable animal model that not only mirrors the pathophysiology of human onychomycosis but is also feasible. Therefore, we attempted to establish an animal model of onychomycosis using immunosuppressed guinea pigs and elucidate the pathophysiology of human onychomycosis. In the present study, we applied Trichophyton mentagrophytes TIMM2789 to the hind limb nails of corticosteroid-treated guinea pigs. The nails were examined macroscopically and histopathologically at 0, 14, and 42 days after a 2-week exposure period to the fungus. A large portion of the experimentally infected nails showed discoloration, which is an important clinical sign, and most infections were confirmed histopathologically in the deep layer of the nail plate at all time points. The infection rates at 0, 14, and 42 days after exposure were 39%, 61%, and 78%, respectively. Thus, we established an animal model of onychomycosis with good reproducibility and that might be appropriate for extrapolation to the pathophysiology of the human disease.