Azole anitifungal drugs such as fluconazole inhibit 14alpha-demethylase. The mechanism of fluconazole action on the plasma membrane is assumed to be ergosterol depletion and accumulation of a toxic sterol, 14alpha-methyl-3,6-diol, that differs in C-6 hydroxylation, B-ring saturation, C-14 methylation, and side-chain modification. Nevertheless, little is known about how these sterol modifications mechanically influence membrane properties and hence fungal viability. Employing time-resolved measurement with a fluorescence anisotropy probe, 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), we demonstrated that fluconazole administration decreased the rigidity of the plasma membrane of Saccharomyces cerevisiae, leading to a dramatic reduction in the order parameter (S) from 0.965 to 0.907 and a 5-fold acceleration of the rotational lipid motion. This suggests that the altered sterol has a deleterious impact on membrane packing, resulting in increased fluidity. Deletion of ERG3 confers hyperresistance to fluconazole by circumventing the accumulation of 14alpha-methyl-3,6-diol and instead produces 14alpha-methylfecosterol lacking the 6-OH group. We found that ERG3 deletion mitigated the fluconazole-induced loss of membrane rigidity with S remaining at a higher value (=0.922), which could contribute to the fluconazole resistance in the erg3Delta mutant. The reduced ability of the 6-OH sterol to stiffen lipid bilayers was supported by the finding that 30 mol % of 6alpha-hydroxy-5alpha-cholestanol marginally increased the S value of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes, while cholesterol and dihydrocholesterol markedly increased it. The decay of the TMA-DPH fluorescence was bimodal in the wild-type strain. This heterogeneity could have arisen from varying degrees of water penetration into the plasma membrane. Fluconazole eliminated the heterogeneity of the dielectric characteristic of the membrane interfacial region, and concomitantly the TMA-DPH lifetime was shortened. Therefore, we conclude that 14alpha-methyl-3,6-diol is insufficient to pack the plasma membrane, allowing water penetration, which is consistent with membrane disorder after fluconazole administration. Our findings illustrate the role of ergosterol in maintaining membrane heterogeneity and preventing water penetration as well as maintaining the rigidity of the plasma membrane interfacial region.