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Dermatophytes are keratinophilic fungi that cause superficial infections of the skin, hair, and nails. The emergence of recalcitrant infections caused by Trichophyton mentagrophytes complex and T. indotineae, particularly in diabetic patients, has heightened the need for reproducible small-animal models to study disease pathogenesis and test antifungal therapies. Here, we describe a diabetic mouse model of dermatophytosis that faithfully mimics human infection. The protocol details (1) preparation of dermatophyte conidial inocula from laboratory or clinical isolates, (2) pharmacological induction of diabetes or immunosuppression in mice, (3) standardized infection of abraded dorsal skin, and (4) downstream assessments of fungal burden and pathology, including clinical evaluation and scoring, enumeration of colony-forming units, and histopathology. Critical parameters, troubleshooting guidance, and anticipated results are provided to ensure reproducibility. This system has been successfully applied to evaluate conventional and investigational antifungal drugs and can be readily adapted for emerging resistant species such as T. indotineae. By integrating diabetic predisposition with current epidemiological trends, this model provides a clinically relevant and cost-effective platform for dermatophyte research and antifungal development. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Preparation of dermatophyte inoculum Basic Protocol 2: Establishment of immunosuppressed or diabetic mouse models of dermatophytosis.