Abstract: Plant pathogenic fungi are one of the important factors leading to crop yield loss and quality decline. Therefore, the development of low-toxicity and high-efficiency antifungal agents has been a research hotspot. In this study, one endophytic fungus was isolated from the stems of Thymus mongolicus Ronn plants and identified as Trichoderma samuelsii by morphological observation and molecular biology assays. The plate antagonistic test showed that the antagonistic activity of T. samuelsii 2-63 strain against eight pathogens reached more than 70.0%, and the highest antagonistic activity against Alternaria alternata that causes black mold of goji berry was up to 87.1%. The plate co-culture experiment showed that the volatile organic compounds (VOCs) of T. samuelsii 2-63 strain also showed different degrees of antifungal activity against pathogens, and the inhibition rate ranged from 31.0% to 68.5%. However, the antifungal activity of T. samuelsii 2-63 strain liquid fermentation broth and the crude extract was low. The combined results of antifungal activity and gas chromatography-mass spectrometry(GC-MS) analysis showed that the key antifungal ingredients of T.samuelsii 2-63 strain were VOCs, and the main component of VOCs was 6-pentyl-2H-pyran-2-one (6-PP). The exploration experiment of 6-PP production showed that rice medium was more conducive to the production of 6-PP than barley medium under solid fermentation conditions, and the maximum yield was 38.6 mg/mL. Under shallow liquid culture conditions, 6-PP first increased and then decreased with the increase of culture time, and the maximum yield was 0.1 mg/mL on the 15^th day of culture. The results provide a theoretical reference and experimental basis for the innovation of microbial agents and pesticides based on Trichoderma.