Polyphenol-rich fraction from edible mushroom, Gomphus floccosus, 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 and total antioxidant capacity. Findings showed that EC 50 values were below 1 mg /ml. Bioactive components namely, total phenols, flavonoids, β-carotene and lycopene were quantified. Estimated putative antioxidant components are in order of phenols > flavonoids > β-carotene > lycopene. High Performance Liquid Chromatography (HPLC) was performed to identify the phenols in the fraction, which detected phenolic acids, myricetin and pyrogallol. Result implies that G. floccosus can be a potential source of natural antioxidants which may be used as food supplement to treat various oxidative stress related diseases.