Invasive fungal infections, such as invasive candidiasis and invasive aspergillosis, have an important impact on morbidity and mortality in intensive care unit (ICU), cancer, and transplant patient populations. New therapies are required to overcome the limitations of the current antifungal armamentarium and the emergence of resistance. The heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that has engendered considerable interest as a potential target for novel cancer and antimicrobial therapies. Fungal Hsp90 was identified as a key regulator of antifungal resistance to both azole and echinocandin antifungals, with distinct features in the two major fungal pathogens, the yeast Candida albicans and the mold Aspergillus fumigatus. This review aims to provide a comprehensive summary on the role of Hsp90 in essential traits of fungal virulence, such as growth, development, stress adaptation and antifungal resistance, as well as the challenge of targeting this highly conserved protein to develop new antifungal strategies.