Abstract: The yield of total alkaloids from stems of Actinidia arguta (A. arguta), Actinidia polygama (A. polygama), and Actinidia kolomikta (A. kolomikta) was used as an index, and the optimal extraction conditions of liquid-solid ratio, ultrasonic power, ultrasonic time, and ethanol concentration were investigated. The inhibitory effects of total alkaloids from the stems of A. arguta, A. polygama, and A. kolomikta against Botrytis cinerea (BC), Colletotrichum gloeosporioides (CG), Alternaria alternata (AA), Corynespora cassiicola (CC) and Pseudomonas syringae pv. Actinidiae (Psa) were investigated. Results showed that, the optimal extraction conditions were an ethanol concentration of 92%, liquid-solid ratio of 24 mL/g, ultrasonic power of 300 W, and an ultrasonic time of 20 min. Under optimized conditions, the highest yields of total alkaloids were 0.41, 0.43 and 0.39 mg/g for A. arguta, A. polygama and A. kolomikta stems, respectively. The total alkaloids from A. arguta stems showed antibacterial activity against Psa, and its minimum inhibitory concentration (MIC) was 50 mg/L. The total alkaloids from the stems of A. polygama and A. kolomikta did not show antibacterial activity against Psa and those from A. arguta, A. polygama and A. kolomikta stems showed no antibacterial activity against BC, CG, AA, and CC. The results confirmed that all three Actinidia spp. stems contained alkaloids and showed inhibitory activity against Psa, providing a theoretical basis for subsequent in-depth studies on the total alkaloids of Actinidia spp.