Lichen is well-known for its various purposes. However, understanding the chemical composition and antimicrobial characteristics of Graphis cf. handelii remains insufficient. In this study, a new compound, graphinone A (1), together with three known compounds, handelone (2), 4-O-methylhiascic acid (3), and ethyl orsellinate (4) were isolated and structurally elucidated. Their chemical structures were established using comprehensive spectroscopic data (1D- and 2D-NMR and HRESIMS). Compounds 1–4 were tested for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and evaluated for the alpha-glucosidase inhibition. Keywords: Graphis cf. handelii; cultured lichen mycobiont; graphinone A; handelone; antimicrobial activity.