The antioxidant activity of the methanol extracts of fruiting bodies of two basidiomycetous mushroom species (Cyclocybe cylindracea and Leccinum duriusculum) was evaluated by high-performance thin-layer chromatography (HPTLC) combined with 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and videodensitometry. The HPTLC separation was achieved on a silica gel layer with acetonitrile–water–acetic acid (75:25:3, V/V). The results were compared with those obtained by the conventional microplate-based DPPH assay. The two methods provided similar results showing that the extract of the C. cylindracea had higher total antioxidant activity than the extract of the L. duriusculum, and the antioxidant activities of both extracts were much weaker than the antioxidant activities of ascorbic acid or gallic acid. Mushroom components in one zone of L. duriusculum extract and three zones of C. cylindracea extract assigned using HPTLC–DPPH–videodensitometry were mainly responsible for the antioxidant activity. Based on a previous study the β-carboline alkaloid C1-S diastereomer of brunnein B in C. cylindracea at hRF 49 contributed to the antioxidant effect the most, which corresponded to 52.0 ± 3.1 ng of ascorbic acid equivalent and 51.7 ± 3.7 ng of gallic acid equivalent. © The Author(s) 2023.