Due to the limited number of antifungals and the emergence of resistance, new therapies against invasive aspergillosis are needed. We show that calcineurin inhibitors are active in vitro against both azole- and echinocandin-resistant Aspergillus fumigatus strains. The heat shock protein 90 (Hsp90) inhibitor geldanamycin had modest activity when used alone, but its combination with caspofungin or tacrolimus (FK506) resulted in fungicidal activity against azole-resistant strains. Targeting the Hsp90-calcineurin axis is a promising alternative strategy against azole-resistant A. fumigatus strains.