Fluconazole (FLC) is a well-known fungistatic agent that inhibits ergosterol biosynthesis. We showed that FLC exhibits dose-dependent fungicidal activity, and investigated the fungicidal mechanism of FLC on Candida albicans. To confirm the relationship between fungicidal activity and the inhibition of ergosterol, we assessed membrane dysfunctions via propidium iodide influx and potassium leakage, as well as morphological change. Interestingly, while membrane disruption was not observed at all tested concentrations of FLC, potassium efflux and cell shrinkage were observed at high dosages of FLC (HDF). Low-dosage FLC (LDF) treatment did not induce significant changes. Next, we examined whether the fungicidal activity of FLC was associated with apoptosis in C. albicans. FLC caused dose-dependent apoptotic responses, including phosphatidylserine externalization and DNA fragmentation. It was also involved in glutathione depletion followed by oxidative damage. In particular, unlike LDF, HDF leads to the disruption of mitochondrial homeostasis, including mitochondrial membrane depolarization and accumulation of calcium and reactive oxygen species. HDF-induced mitochondrial dysfunction promoted the release of cytochrome c from mitochondria to the cytosol, and activated intracellular metacaspase. In conclusion, the dose-dependent fungicidal activity of FLC was due to an apoptotic response in C. albicans.