This study aims to investigate the antifungal secondary metabolites from the endophytic fungus Aspergillus sp. FH-2 isolated from Valeriana officinalis L., cultured on a rice medium. Based on the one strain many compounds (OSMAC) strategy, the optimal culture medium was first screened to facilitate large-scale fermentation. The metabolites were then isolated and purified using a combination of silica gel chromatography, ODS column chromatography, and semi-preparative liquid chromatography. Structural elucidation of the obtained compounds was carried out by comprehensive analysis of their physicochemical properties and spectroscopic data, including NMR, IR, and MS. Eight compounds were isolated from the ethyl acetate extract of the Aspergillus sp. FH-2 strain. These were identified as: asperalipha A (1), questin (2), 6,8-dihydroxy-3-methylisocoumarin (3), 3,6,8-trihydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin (4), 5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone (5), 7-drimen-9α,11,12-triol (6), methyl indole-3-carboxylate (7), and N-[2-(4-hydroxyphenyl)ethenyl] formamide (8). Among these, compound 1 was identified as a new compound. The antifungal activities of all compounds against Colletotrichum gloeosporioides were determined using the mycelial growth rate method. The results indicated that compound 1 exhibited superior inhibitory efficacy compared to the positive control carbendazim. Compounds 2 and 4 also demonstrated moderate inhibition, with EC₅₀ values ranging from 43.12 to 79.04 μg/mL. Molecular docking results revealed that compound 1 possesses a stronger binding affinity to the target protein than the positive control drug (carbendazim), with a binding energy of -6.30 kcal/mol.