Pleurotus ostreatus is an edible mushroom with pharmacological potential, due to its metabolites, mainly polysaccharides. On here, the development of a new methodology for the recovery of extract rich in antioxidant polysaccharide was reported. The extracts were characterized, evaluated for antioxidant activity in vitro and in cell models and cytotoxicity. The best defined extraction condition was 25 MPa, 433.15 K, and 20% HO, with 30.69% of the total yield and 0.921 mg of CHO. The anomeric bonds, identified in the FTIR and NMR spectrum, indicate that the extracts are a mixture of heteropolysaccharides, β-glucans, α-glucans, and oligosaccharides. The best extraction condition has 80.83% of antioxidant activity, without cytotoxic effect in vitro. In addition to antioxidant activity in cell model, increasing protection against oxidative damage induced by HO. Finally, HO + CO-SFE technology can be used to obtain extracts rich in antioxidant polysaccharides with pharmacological and food potential.