The antibacterial agent mupirocin (pseudomonic acid A) is used as a topical agent in the treatment of superficial infections by Gram-positive bacteria, particularly Staphylococcus aureus. However, we demonstrate here that the compound also inhibits the growth of a number of pathogenic fungi in vitro, including a range of dermatophytes and Pityrosporum spp. It inhibited the incorporation of amino acids and precursors of RNA, but not that of acetate, by Trichophyton mentagrophytes. It also inhibited the isoleucyl-tRNA synthetase from Candida albicans, indicating a mechanism of action similar to that in bacteria. When administered topically, mupirocin was efficacious in a T. mentagrophytes ringworm model in guinea pigs. These results suggest that mupirocin could have clinical utility for superficial infections caused by dermatophytes.