The treatment of dermatophytosis has improved considerably over the past several decades following the introduction of the oral antifungals such as azoles and amphotericin B. However, these drugs have had limited success because the treated fungi often develop drug resistance, resulting in recurrence when applied in various topical formulations. Thus, there are constant needs for new topical agents that are effective against dermatophytosis. Dipyrithione is an attractive candidate to become an antifungal agent due to its broad spectrum of antimicrobial activities. In this study, we determined that dipyrithione could potently inhibit the growth of Trichophyton rubrum, which is the most common cause of dermatophytosis. The MIC value of dipyrithione against T. rubrum was measured as 6.03 μM, as compared with miconazole (MIC: 1.38 μM). Additionally, the compound caused morphological changes in the fungi, which was examined using the morphological interference assay. The in vivo experiment further revealed that dipyrithione had a healing effect on the skin of guinea pigs infected with T. rubrum. Our studies have demonstrated that dipyrithione had a potent antifungal activity in vitro and in vivo, suggesting that it could be formulated as a potential antifungal lead compound in search for novel therapeutic agents against dermatophytosis.