Metabolites of Endophytic fungi as an important resource to find new bioactive compounds has received growing concern. In this paper, 30 endophytic fungi strains were isolated from roots, stems and leaves of Agrimonia pilosa Ledeb respectively, and morphological observation was employed to identify the endophytic fungi strains. The results showed that 30 endophytic fungi strains from Agrimonia pilosa Ledeb were Penicillium, Acremonium,Fusarium, Rhizopus, Saccharomyces, Arachniotus Schrot, Mucor, andSclerotinia. Meantime,effects of these Endophytic fungi strains on the growth of Staphylococcus aureus were investigated. The result indicated that 9 strains exerted antibacterial activity. Then, extracts of Endophytic fungi fermentation were used to treat Staphylococcus aureus, and the result indicated that only 3 strains of Fusarium exerted the antibacterial activity. Our results might be significant to study the bioactivity of endophytic fungi from Agrimonia pilosa Ledeb and find bioactive compounds in future.