This retrospective study of 371 cats treated for dermatophytosis sought to determine how often a first negative fungal culture was indicative of mycological cure as compared with 2 negative fungal cultures. Results demonstrated that a first negative fungal culture for Microsporum canis indicated mycological cure in 90.3% of cats, and subsequent cultures remained negative. Of the remaining cats, other than being lesional, 19 were healthy and had 1 negative fungal culture within the first 3 weeks of treatment, followed by ≥1 positive fungal cultures; these cats went on to mycological cure without event. Another 17 cats had concurrent medical illness in addition to dermatophytosis. These patients initially had resolution of lesions and negative fungal cultures but ≥1 positive fungal culture during treatment; mycological cure was not achieved until the concurrent illness was resolved. In otherwise healthy cats in which high treatment compliance is achieved, 1 negative fungal culture may be sufficient to indicate mycological cure.