Abstract BACKGROUND Despite the revelation of numerous uncharacterized biosynthetic gene clusters (BGCs) in the termite‐associated fungus Aspergillus chevalieri BYST01 through genome mining, no suitable culture conditions have been established to activate the production of new secondary metabolites. RESULTS Herein, using the OSMAC (one strain many compounds) strategy to preliminarily analyze the potential metabolites of A. chevalieri BYST01. A total of 23 metabolites were isolated and identified from minimal medium cultures in A. chevalieri BYST01, including one new compound ( 2 ) and 22 known compounds ( 1 , 3 – 23 ), which were categorized into seven distinct skeletal types. Partial metabolites were evaluated for their antibacterial and phytotoxic activities. Pannorin C ( 1 ) exhibited strong antibacterial activities against Staphylococcus aureus , Pseudomonas syringae pv. actinidae , Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc). Citreorosein ( 18 ) and stemphyperylenol ( 19 ) showed excellent antibacterial activity against Xoc with inhibition zone diameters (IZD) of 15.5 and 19.6 mm, respectively. Notably, dihydroauroglaucin ( 6 ), flavoglaucin ( 9 ), and compound 18 demonstrated significant protective efficacy against rice bacterial blight at 0.5 μg mL −1 , with protection rates of 84.82, 82.68, and 82.88%, respectively, which was stronger than that of commercial bactericide kasugamycin (81.52%). Additionally, compound 19 and vivotoxin II ( 23 ) exhibited strong inhibitory effects against the root growth of the Echinochloa crusgalli with an inhibition rate of 67.6 and 87.6% at the concentration of 100 μg mL −1 , respectively. Tenuazonic acid ( 22 ) and vivotoxin II ( 23 ) significantly inhibited the root growth of Abutilon theophrasti at the concentration of 100 μg mL −1 (82.5 and 100%, respectively). The possible herbicidal mechanisms of compounds 19 , 22 , and 23 were preliminarily investigated by molecular docking simulation. CONCLUSION These findings validate OSMAC for uncovering bioactive metabolites from BYST01 and provide lead compounds for the development of bioactive drugs in food and agricultural fields. © 2025 Society of Chemical Industry.