Objective To determine the classification status of endophytic fungus Eef-10. To isolate and identify secondary metabolites of Eef-10 and evaluate their antibacterial and antitumor activities in order to obtain natural active compounds.Method The endophytic fungus was identified by combining morphology and molecular biology methods. The secondary metabolites were mainly separated and purified by vacuum silica gel column chromatography, sephadex LH-20 column chromatography and semi-preparative HPLC. The compounds were identified mainly based on ¹H NMR and ¹³C NMR spectral data as well as related references. The antibacterial activities against five different test bacteria were determined by the MTT method and the antitumor activities against two cancer cells were determined by the CCK8 method.Result Three compounds were isolated from the endophytic fungus Eef-10, namely atraric acid (Compound Ⅰ), ethyl 2, 4-dihydroxy-3, 6-dimethylbenzoate (Compound Ⅱ) and 4-methyl-5, 6-dihydro-2 H-pyran-2-one (Compound Ⅲ). Compound Ⅱ displayed strong inhibitory activities against five test gram-negative bacteria, and the IC₅₀ values were 35.87-55.50 μg/mL. The IC₅₀ values of compound Ⅰ were 67.25-130.55 μg/mL, while compound Ⅲ had IC₅₀ values of more than 200 μg/mL for all five bacteria. The IC₅₀ value of compound Ⅱ for Hep-G2 was 1.50 μg/mL, which was stronger than the positive control camptothecin of 3.6 μg/mL.Conclusion Compounds Ⅰ-Ⅲ were isolated from the endophytic fungus Chaetomium sp. Eef-10 and compound Ⅱ showed great antibacterial and antitumor activities on R. solanacearum and Hep-G2 tumor cells.