Abstract The aim of this study was to isolate the endophytic fungi from Sophora flavescens and define its antimicrobial action and structure. The effects of this active substance on soil microorganism circumstances were also conducted. Column chromatography and freeze drying were used to isolate and purify the antimicrobial substance. TLC biological autoradiography was applied to trace. HPLC method was employed to measure the purity. Analysis of the structure used 1H-NMR, 13C-NMR and LC-MS methods. Plate method was applied to measure the effect on microorganism circumstances. The results showed that after isolating and purifying the fermentation liquor of BS001, the structure of bacteriostatic active composition was 6,7-(2′E)dibutenyl-5,8-dihydroxy-(Z)-cyclooct-2-ene-1,4-dione, which was identified by spectroscopy. The substance could increase the number of bacteria and fungi while decreased the number of antinomies in soil. A new antimicrobial substance 6,7-(2′E)dibutenyl-5,8 -dihydroxy- (Z)- cyclooct - 2- ene-1,4-dione was extracted from fermenting liquor of BS001 which was an endophytic fungi of Sophora flavescens. It could promote the beneficial flora but detrimental flora. This conclusion provides exploiture foreground for biopharmaceuticals and biopesticide.