Abstract: Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeasts. We previously demonstrated that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species (ROS), pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1-, sur1-, skn1- and erg3-deletion mutants, are miconazole resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft disturbing compounds on miconazole antifungal activity as well as on the miconazole-induced actin cytoskeleton stabilization and ROS accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts on miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.