Background: The effect of substrate and strain on the nutritional and functional properties of mushroom flours and protein concentrates (PCs) has not thoroughly been investigated. Methods: The proteins of P. ostreatus flours (strains AMRL144 and 150) were isolated following alkaline extraction (pH 12) and isoelectric precipitation (pH 4) as it was determined by the solubility curves. The protein quality of the flours and PCs were evaluated by determining the protein solubility index (PSI) and their functional properties, such as water (WAC) and oil absorption capacity (OAC), foam capacity (FC), foam stability (FS) and emulsion stability (ES). The amino acid (AA) composition of the flours was determined by reversed-phase liquid chromatography after protein hydrolysis and o-phthalaldehyde derivatization. Results: The PSIs of the flours and PCs of P. ostreatus were comparable to those of soy protein flours and protein concentrates. The highest AA concentration was found for both strains when cultivated on the barley and oat straw (BOS) substrate, showing a similar trend as the protein content. A principal component analysis (PCA) indicated an impact of the strain on the functional properties. Conclusions: Both strains can produce high quality proteins especially when cultivated on wheat straw (WS). The FS was positively correlated with the P. ostreatus strain AMRL150 whereas the FC was positively correlated with AMRL144.