Effect-directed isolation of free radical scavengers from the methanol extract of the freeze-dried fruiting bodies of the cultivated basidiomycetous mushroom, black poplar (Cyclocybe cylindracea), yielded a β-carboline alkaloid. Its structure was determined based on ESI-TOF-MS/MS, NMR and circular dichroism spectra by comparison with published data. The compound, identified as the C1-S diastereomer of brunnein B, exhibited explicit radical scavenging activity (EC50 = 119.1 ± 1.2 μg/mL). The quantity of the active component was determined with HPLC-MS in the fruiting body (36.2 ± 2.8 ng/g DW, dry weight) and its different tissues such as peel (94.7 ± 1.9 ng/g DW), inner cap (90.5 ± 1.3 ng/g DW), gills (71.5 ± 0.6 ng/g DW), and stipe (162.2 ± 1.7 ng/g DW). It is a β-carboline alkaloid that was not reported previously. © 2019 Elsevier B.V.