Polyphenol-rich fraction from edible mushroom, Pleurotus flabellatus, was tested for in vitro antioxidant activity in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and chelating effect of ferrous ions, reducing power, total antioxidant capacity assay and estimation of bioactive components namely, total phenol, flavonoid, β- carotene, lycopene and ascorbic acid. Findings showed that EC50 values were below 1 mg /ml. Estimated putative antioxidant components are in order of phenol > flavonoids > ascorbic acid > β-carotene > lycopene. Result implies that P. flabellatus can be a potential source of natural antioxidant which may be used as food supplement to treat various oxidative stress related diseases.