Green synthesis of Zinc oxide nanoparticles using biological sources is of great interest in nanotechnology field owing to its stability and biocompatible properties. The present study was undertaken to synthesize ZnO NPs using aqueous extract of Agaricus bisporus and to evaluate its in-vitro biological effects. The biosynthesized ZnO NPs were characterized by various spectroscopic techniques. Zeta potential (-20.5mV) confirmed the stability of the nanoparticles. SEM and TEM analyses identified spheriod shaped sturctures of ZnO NPs. Antibacterial activity by agar well diffusion method indicated higher zone of inhibition against gram+ve bacteria (B.subtilis (32mm±1.05); S.aureus (28mm±1.1); E.faecalis (21mm±1.05)) than gram-ve bacteria (P.vulgaris (22mm±0.9); K.pneumoniae (18mm±0.85); E.coli (16mm±0.8)). Antioxidant assay proved that ZnO NPs exerts maximum radical quenching effects against DPPH (91 %) then ABTS (86 %) and NO (81 %) at 50μg/mL respectively. MTT assay showed cytotoxicity of the ZnO NPs against SW 620 cell lines (human colon cancer). © 2020 Elsevier Ltd.