Abstract: In this present paper, the optimization of microwave extraction (ME) of Cremastra appendiculata (D. Don) Makino was performed using the single factor design and response surface methodology. The ME extraction yield along with the contents of three major components of C. appendiculata Makino, including militarine, dactylorhin A, and colchicine was selected as the comprehensive indicators to evaluate the extraction process. Subsequently, the HPLC fingerprint of the above three components from 13 batches of C. appendiculata Makino was investigated. Finally, the anti-fungal activity of C. appendiculata Makino extract was evaluated by conducting a disk-diffusion method. The results demonstrated that the optimal ME technologies of C. appendiculata Makino were the ethanol concentration of 55%, the solid-liquid ratio of 1:30 g/mL, and the extraction time of 17 min. Under the above conditions, the extract yield was 12.83%. HPLC fingerprint analysis results showed that the contents of three major components displayed a consistent trend in all 13 tested samples. Militarine was found existed in 13 batches of C. appendiculata Makino, while dactylorhin A and colchicine were not detected in one sample. The results indicated a content difference in three active ingredients in C. appendiculata Makino from different areas. The anti-phytopathogenic activity results showed that the C. appendiculata Makino extract did not shown inhibitory activity against Colletotrichum gloeosporioides, Phytophthora capsica, Botrytis cinerea, Alternaria solani, Pseudomonas fluorescens, Xanthomnas oryzae, and Fusarium graminearum with their inhibition zones were less than 6 mm. However, the C. appendiculata Makino extract exhibited inhibitory activity toward to Ralstonia solanacearum with the diameter of the inhibition zone was 8.5 mm, showed antibacterial selectivity. In this study, the optimal ME technologies of C. appendiculata Makino was determined, and the established method the contents of three major components of C. appendiculata Makino was stable and feasible.These results can provide the basis for the quality control and comprehen-sive utilization of C. appendiculata Makinos.