Mushrooms comprise a vast source of new pharmaceutical and nutraceutical products. Submerged fermentation of the mycelial form of mushroom-producing fungi is promising for efficient production of their biomass and active metabolites. The aim of this study was the isolation and identification of bioactive metabolites derived from the edible mushroom Pleurotus ostreatus biomass, produced by submerged fermentation in a batch stirred tank bioreactor. The dichloromethane extract was fractioned by medium pressure liquid chromatography (MPLC) and Sephadex LH-20 column chromatography. For extracting efficiently the phenolic compounds of the methanolic extract, an adsorption-desorption process, using XAD-4 type resin, was performed. The pure compounds were elucidated with 1D/2D NMR-spectroscopic analyses, NMR data comparisons, and chemical correlations combined with GC/MS- LC/MS experiments. The compounds afforded by the dichloromethane extract were identified as linoleic acid (1), oleic acid (2), stearic acid (3), palmitic acid (4) and their corresponding methyl esters (5-8, respectively), benzoic acid (9), trans 3, 4-dihydro-3, 4, 8-trihydroxynapthalen-1(2H)-one (10), 4-hydroxy- benzaldehyde (11), indolo-3-carboxylic acid (12) and uracil (13). The investigation of the methanolic extract led to the isolation of 3-formyl-pyrrole (14), 4-hydroxy-benzoic acid (15), uridine (16), nicotinic acid (17) and nicotinamide (18). Based on existing literature data, all these compounds exhibit valuable biological properties. The information obtained is considered fundamental for further investigation of the P. ostreatus fermentation process on an industrial scale for enhanced bioactive metabolite production.