An efficient microwave-assisted steam distillation followed by a solvent extraction method (MSDE) was established to isolate the essential oil, asarinin and sesamin from the roots and rhizomes of Asarum heterotropoides var. mandshuricum (AHVM). In the MSDE technology applied in this work, moistened roots and rhizomes of AHVM powders are applied to this system. After extracting the essential oil by steam distillation, the powder residue of the roots and rhizomes of AHVM is relatively dry. Hence, this method can save energy consumption, and the loss of asarinin and sesamin can be reduced during the subsequent extraction process and improve essential oil yield. The developed method extracted the essential oil to reach the maximum extraction amount (15.80 ± 0.79 mL/kg) after microwave irradiation for 30 min and maintained the yield balance. Through comparison, MSDE was chosen as the best approach, and under the optimum conditions (90 % aqueous ethanol, liquid–solid ratio of 19.0 mL/g and microwave irradiation power of 332 W) obtained by first order kinetic models and response surface analysis with Box–Behnken design, satisfactory yields of asarinin (1.81 ± 0.72 mg/g) and sesamin (0.74 ± 0.09 mg/g) were achieved. In accordance with the yields of asarinin and sesamin, our research optimized the process parameters. According to the comprehensive comparison of these parameters, the proposed process described in this study has greater advantages than traditional methods. Minimum inhibitory concentrations (MICS) of the essential oil, which obtain from the roots and rhizomes of AHVM of five kinds of Fusariums spp. (F. avenaceum, F. trichothecioides, F. sporotrioides, F. sambucinum and F. culmorum) were determination to be 1.25 mg/mL, 1.5 mg/mL, 1.5 mg/mL, > 2 mg/mL and > 2 mg/mL, respectively. The results of the antifungal test showed that the essential oil from the roots and rhizomes of AHVM showed remarkable antifungal activities, in which, F. avenaceum was the most sensitive to the essential oil, with the MIC50 values of 0.625 mg/mL.