Antioxidant property of natural substances especially polyphenols is of current concern as it can protect human health from various chronic diseases. In this context, mushrooms have repeatedly been informed as a reservoir of several bioactive substances; although they still represent an almost untapped group. One such less explored fungus is Macrocybe lobayensis that has indigenously been accepted as gastronomic delicacy along different states of India. Thus, the present work was designed for the determination of free radical scavenging activity of polyphenol-rich fraction isolated from the taxon and for this purpose, a quite different extraction process has been followed to maximize yield. Initially, the formulation was characterized quantitatively and qualitatively to estimate therapeutic substances. Further, the extract was subjected to seven in vitro model systems to visualize the magnitude of antioxidant property. The fraction was found to be an assemblage of various components like phenol (pyrogallol>cinnamic acid> ferulic acid), flavonoid, ascorbic acid, Β-carotene and lycopene with high recovery percentage. Besides, the formulation was detected to be extremely powerful for scavenging hydroxyl radical, followed by DPPH, ABTS and superoxide radicals. The fraction was also able to chelate metal ion efficiently while the reducing power was found to be moderate in action. Overall, the present study implies that M. lobayensis could be used in food; cosmetics, as well as pharmaceutical industry and in that note, the extracting method described herein, can be followed to prepare a fraction with high yield and strong antioxidant property. © 2018 Pharmascope Publications. All rights reserved.