Abstract: The aim of this study was to obtain effective substances from plant against Clavibacter michiganensis subsp. michiganensis. On the basis of single factor experiments,an interactive orthogonal experiment L₂₇(3¹³)was designed. Antibacterial activity was used to optimize the extraction conditions of active substances from Athyrium sinense against C. michiganensis subsp.michiganensis. The active substances obtained under the optimal conditions were used to determine the three indexes of active oxygen,intracellular protein contents and extracellular protein contents in C. michiganensis subsp. michiganensis. The results showed that material to liquid ratio and temperature had extremely significant effects on the extraction of antibacterial active substances,and the interaction between them had a significant effect. The optimal extraction conditions were determined as material to liquid ratio 1:10 g/mL,extraction temperature 80 ℃ and extraction time 8 h,and the diameter of the inhibition zone was 27.33 mm. After treatment with A. sinense active substance,the levels of intracellular reactive oxygen species and extracellular protein content increased,and the intracellular protein content decreased. According to the results,the active substance of A. sinense against C. michiganensis subsp.michiganensis can be obtained,which should provide a new idea for the control of this pathogen.