Aim: To investigate the antifungal mechanism of clioquinol and indicate that clioquinol has potential as a novel therapeutic antifungal agent.Materials & methods: Analyze differentially expressed genes of Candida albicans treated with clioquinol using RNA-sequencing. The effects on cell wall and membrane features, virulence factors, apoptosis-induced cell death were also investigated.Results: The differentially expressed genes of C. albicans after treated with clioquinol focused on cell wall and membrane synthesis, antioxidant system and energy metabolism. Clioquinol did not change cell wall components levels while it decreased squalene epoxidase activity to influence the ergosterol biosynthesis in cell membrane. It also decreased cellular surface hydrophobicity and induced β-glucan unmasking to attenuate virulence factors. Meanwhile, clioquinol influenced enzyme activities involved in antioxidant system, citrate cycle, oxidative phosphorylation and decreased the ATP levels. Clioquinol induced apoptosis in C. albicans to exert its fungicidal activity. It induced reactive oxygen species and calcium ion elevation, leading to loss of mitochondrial membrane potential, cytochrome C release, metacaspase activation, thereby triggering apoptosis.Conclusion: Clioquinol exerted anti-C. albicans activity through influencing cell membrane, attenuating virulence factors and inducing apoptosis.