The cultured mycobiont of the Vietnamese crustose lichen Diorygma sp. was subjected to chemical investigation, which led to the isolation and characterization of a new guaiane sesquiterpene, diorygmone F (1), along with three known compounds, pruinosone, β-sitosterol, and methyl 4-hydroxybenzoate. Structural elucidation was achieved through extensive spectroscopic methods, including NMR, HR-ESI-MS, and ECD. Compound 1 displayed modest inhibitory activity against α-glucosidase, with an IC50 value of 131 ± 1.4 μM, and showed no cytotoxicity against the HepG2 cancer cell line. Keywords: cultured lichen mycobiont, Diorygma sp., guaiane sesquiterpene, α-glucosidase inhibition, cytotoxicity.