Biofabrication of Copper nanoparticles (CuNPs) using Agaricus bisporus aqueous extract and its potential in-vitro biological activities were attempted in the present work. The synthesized CuNPs was confirmed by absorption peak at 551 nm using UV–Vis spectroscopy. The crystalline properties and the biofunctional groups involved in the reduction process were authenticated by XRD and FTIR spectroscopic techniques. Zeta potential value of −40 mV confirmed the stability of CuNPs. SEM and TEM analysis revealed agglomerated morphology and the presence of elemental Cu was confirmed by EDAX. In-vitro anti-bacterial assay demonstrated good bactericidal effects with more inhibitory action against Enterobacter aerogens (15 ± 0.08 mm). Anti-oxidant assays revealed potential scavenging effect against ABTS radical (82%); NO radical (76%) and DPPH radical (72%). Cytotoxic assay also revealed potential inhibitory effects against human colon cancer cell lines (SW620) with 60% cell viability recorded at 500 μg/ml concentration. Fluorescent microscopic observation of SW620 cells stained by DCFHDA and acridine orange dye displayed fragmentation, membrane loss and generation of ROS apoptotic features. © 2020 Elsevier B.V.