Objective: This study aimed to optimize the microwave-assisted water distillation extraction process of citronella volatile oil, and to evaluate the antioxidant properties and antifungal ability of citronella volatile oil. Methods: The Box-Behnken response surface method was used to analyze the influence of liquid-to-material ratio, microwave power and microwave time on the yield of volatile oil. Gas chromatography-mass spectrometry (GC-MS) analysis was used to identify the composition of the volatile oil. Thereafter, DPPH· and ABTS· scavenging methods and β-carotene bleaching experiment were utilized to evaluate the anti-oxidation ability of volatile oil. Perforation method and broth dilution method were used to analyze the anti-fungal ability of volatile oil. Results: The best process parameters of microwave extraction were liquid-to-material ratio 7.185∶1.000 (mL/g), power 700 W, time 21.707 min, and the essential oil yield was 15.965 mL/kg·DW. The citronella volatile oil was found showing good inhibition activity, on the free radicals DPPH·, ABTS· and β-carotene/linolenic oil produced by the acid system, and the 50% inhibitory concentration (IC₅₀) values were (0.546±0.002), (1.694±0.001) and (0.145±0.003) μg/mL, respectively. Citronella volatile oil also showed a relatively obvious inhibitory effect on Pseudocercosis, and its minimum inhibitory concentration (MIC) was 2.5 μg/mL, with the minimum bactericidal concentration (MBC) of 3 μg/mL. Conclusion: Compared with the traditional water distillation method for extracting volatile oil, the microwave-assisted water distillation method is more efficient, and the extracted volatile oil components are more abundant. The excellent biological activity of citronella volatile oil also provides a theoretical basis for the development of antibacterial agents and antioxidants.