Water soluble polysaccharides (WSP) were isolated from Lentinus edodes fruiting bodies. The mushrooms were previously subjected to various processing techniques which included blanching, boiling, and fermenting with lactic acid bacteria. Therefore, the impact of processing on the content and biological activities of WSP was established. Non-processed fruiting bodies contained 10.70 ± 0.09 mg/g fw. Boiling caused ~12% decrease in the amount of WSP, while blanched and fermented mushrooms showed ~6% decline. Fourier transform infrared spectroscopy analysis (FTIR) confirmed the presence of β-glycosidic links, whereas due to size exclusion chromatography 216 kDa and 11 kDa molecules were detected. WSP exhibited antioxidant potential in FRAP (ferric ion reducing antioxidant power) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assays. Cytotoxic properties were determined on MCF-7 and T47D human breast cell lines using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. Both biological activities decreased as the result of boiling and fermenting.