The capability to synthesize the extracellular polysaccharide (EPS) is widespread among eight mushroom species which accumulated 0.6–2.2 g/1 of EPS in submerged cultivation. Glucose, maltose, and mannitol were the most appropriate carbon sources for biomass and EPS production. Organic nitrogen sources appeared to be the most suitable nitrogen sources for biomass and EPS accumulation. The cultivation process in shake flasks was successfully reproduced in a laboratory fermentor with enhanced EPS production. The highest yield of EPS (3.8–4.0 g/1) was achieved in cultivation of Agaricus nevoi and Inonotus levis.