In order to investigate the optimal extraction process of Osmanthus fragrans flavonoids and their antifungal effect on Candida albicans (C. albicans), the following experiments were conducted. The extraction rates of Osmanthus fragrans flavonoids by ultrasonic extraction, ultrasonic-assisted enzyme extraction and ultrasonic-assisted double aqueous phase extraction were compared, the method with the highest extraction rate was selected for process optimization experiments of single factor and response surface; the components of four varieties of Osmanthus fragrans flavonoids were identified by liquid chromatography-mass spectrometry (UPLC-MS/MS). The inhibition effects of four varieties of Osmanthus fragrans flavonoids on C. albicans in terms of minimum inhibitory concentration (MIC), cell membrane integrity and biofilm formation were determined. The results showed that after the single-factor and response surface optimization experiments, the best extraction process of cinnamon flavonoids was 50 ℃, the liquid-solid ratio was 17 mL/g, and the ethanol concentration was 52%, and the best extraction rate were 13.88% for Thunbergii group, 8.79% for Latifolius group, 13.16% for Aurantiacus group, and 10.28% for Semperflorens group under these conditions. Under these conditions, the extraction rates of thunbergii group, latifolius group, aurantiacus group and semperflorens group were 13.88%, 8.79%, 13.16% and 10.28%, respectively. The antifungal effect of different varieties of Osmanthus fragrans flavonoids on C. albicans was different, among which, Osmanthus fragrans var. thunbergii had the best antifungal effect with MIC value of 1.5 mg/mL, which could significantly improve the permeability of the cell membrane of C. albicans within 24 h, leading to the efflux of cell contents, and the inhibition rate of biofilm formation could reach up to 46.59%. Therefore, cinnamon flavonoids can be studied and developed as natural antibacterial agents in depth.