Dermatophytosis is common in cats. Diagnosis usually involves collecting plucked hair from focal lesions and/or sampling the whole body using the sterile tooth-brush method. This study evaluated the best sampling protocol for the tooth-brush method. Two samples were obtained from each of the 26 cats included in the study and used to inoculate agar plates by either pressing the toothbrush into the agar (procedure A) or by transferring collected hair and scales to the culture plate in addition to pressing the toothbrush into the agar (procedure B). Results show that plating the sample without adding hairs and scale yielded better accuracy, as the added hair and scale introduced contaminant molds that competed with Microsporum spp, inhibiting their growth.