This study investigates the variations in the chemical composition of Juniperus chinensis Roxb.'Kaizuka Procumbens' essential oil(JCEO) collected at different harvesting times and locations, as well as the differences in antifungal activity against the soil-borne pathogen Sclerotium rolfsii, and optimizes the extraction process of JCEO. JCEO was extracted using ultrasound-assisted hydrodistillation, and the extraction process was optimized through response surface methodology. The chemical components of the JCEO from different harvesting times and locations were analyzed by GC-MS, and the relative contents of each component were calculated. The antifungal activity of JCEO was evaluated using the mycelial growth inhibition rate method. Orthogonal partial least squares discriminant analysis and correlation analysis were employed to assess the differences in the components and their relationships with antifungal activity. The results showed that the optimal extraction conditions for JCEO were ultrasonic time of 15.6 min, material-liquid ratio of 1∶9.16 (g/mL) , and extraction time of 3.4 h. The 12 JCEOs were detected with 28 common components, and the relatively high contents of the components were sabinene (15.95%-40.77%), borneol acetate (1.27%-20.1%), 2-undecanone (2.01%-12.77%), γ-terpinene (2.44%-10.51%), terpinen-4-ol (2.24%-8.24%), cadinene (1.62%-7.07%), etc. The essential oil exhibited significant activity, with differences observed among the 12 JCEOs. Correlation analysis indicated that sabinene, linalool, and borneol acetate were significantly positively correlated with the antifungal activity of JCEO. This study determined the optimal extraction process for JCEO and compared the differences in essential oils collected at harvesting times and locations, providing a reference for the development and utilization of JCEO.