Mushroom-derived polysaccharides, which are the primary bioactive constituents, are beneficial for human health. Polysaccharides have immuno-modulation, antitumor, and antioxidant properties. Additionally, they have antiviral properties and protect against chronic radiation stress. In this study, high yield water-soluble polysaccharides were obtained from Pleurotus djamor var. roseus basidiocarps. The crude polysaccharide (CP) was extracted sequentially by hot water and ethanol precipitation. The yield of the brown CPs was 5.6% dw. Diethylaminoethyl cellulose and Sepharose-6B column chromatography of CPs generated several fractions. Total glucan content was determined in all the fractions. The F1 fraction displayed the highest sugar content and was considered as a purified polysaccharide (PP). The total glucan and β-glucan content in the four fractions ranged between 76.85-2.95% and 75.08-1.46%, respectively. The yield of the PPs was 300 mg, and it was obtained as a white powder. The PPs were characterized by Fourier-transform infrared spectroscopy (FTIR) and thin-layer chromatography. The FTIR spectral details confirmed the presence of a xylopentose polysaccharide. The antioxidant activity of the PPs was evaluated using in vitro 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay and superoxide radical scavenging assay. The PPs showed strong DPPH free radical and superoxide anion radical scavenging activities in a dose-dependent manner. Purified PPs free of phenolics, protein, and carbohydrates were mainly responsible for the radical scavenging activity. The data suggest the potential of PPs as natural antioxidants.