Many antifungal drugs including the highly successful azoles target the fungal-specific sterol, ergosterol, yet the molecular identity of cellular pathways mediating antifungal activity remained obscure. A recent study on the requirement of ergosterol in vacuolar H+-ATPase (V-ATPase) function uncovered a critical role for ion homeostasis downstream of azole inhibition of ergosterol biosynthesis. Here we review the functional link between regulation of V-ATPase by fungal membrane lipid components and discuss contributions of V-ATPase function to pathogenicity. The importance of pH homeostasis in virulence highlights its potential as target in antifungal chemotherapy.