Many species of higher basidiomycetes have traditionally been used because of their medicinal properties. The positive effects associated to the consumption of those fungi have been mainly attributed to cell wall polysaccharides, which have important structural roles and are present throughout the entire life cycles of fungi. One of the most consumed and studied species native of the Americas is Agaricus subrufescens, a mushroom prescribed in different countries for prophylaxis and noninvasive treatment of numerous health-related disorders. Prior to the process of extraction, purification, and application of these polysaccharides, one needs to be concerned with the preservation of the specimen and production of fungal biomass. Even though basidiomata (syn. fruiting bodies, mushrooms) generally yield larger volumes of biomass when compared to the mycelium, cultivation of mycelium allows a more efficient control of the process and, therefore, is the method of choice of polysaccharide production. Mycelial biomass can be produced by solid-state fermentation (SSF) or submersed fermentation (SmF). Further separation and concentration of bioactive polysaccharides can be done by means of porous membranes, such as tangential flow nanofiltration.