Abstract: An endophytic fungus CWJ01 with good antibacterial activity was isolated from the leaves of Acanthopanax senticosus. In order to improve the antibacterial activity of the fermentation broth of this strain and explore the substance basis of its antibacterial activity, the fermentation conditions of the endophytic fungus CWJ01 were optimized by orthogonal experiment according the antibacterial activity on the basis of single factor experiments. The secondary metabolites contained in the endophytic fungus CWJ01 were separated by column chromatography. The strains of the endophytic fungus CWJ01 were identified by morphology and ITS sequence analysis. The results showed that the optimum fermentation conditions of CWJ01 were as follows: PDB as the base medium, 2% glucose as the carbon source, 1% peptone as the nitrogen source. The initial pH of the medium was 7.0, the liquid content of the medium was 60%, the inoculated strain was 5%, the rotating speed was 140 r/min, the temperature was 28 ℃, and the culture time was 15 days. Under the optimal process conditions, the diameter of inhibition zone of CWJ01 fermentation broth against Staphylococcus aureus was 27.48±0.17 mm. Trans-cinnamic acid and benzoic acid were isolated from the fermentation broth of endogenous fungus CWJ01, and the endophytic fungus CWJ01 was identified as Penicillium polonicum. This study can provide reference for further exploration of antibacterial active substances of endophytes.