Oyster mushroom (Pleurotus ostreatus) is a good source of bioactive compounds that have numerous healthbenefits and medicinal properties. In this study, the effect of P. ostreatus cultivation in agro-waste substrates on itspotential as a food additive with antioxidant and antimicrobial activities was investigated. Extracts of P. ostreatus cultivatedon mixed rice and wheat straws (RS+WS) substrates showed higher total polyphenols (TPC), total flavonoids(TFC),α-tocopherol content, ferric reducing antioxidant power and antibacterial activities than those cultivated on ricestraw (RS) alone. The HPLC analysis of their ethanolic extracts revealed 11 phenolic compounds including p-hydroxybenzoic acid, naringenin, kaempferol and apigenin as major compounds. The aqueous and ethanolic extracts of bothcultivates showed significant antibacterial activity against Salmonella typhimurium, Escherichia coli, and Staphylococcusaureus, while no inhibitory effect on Bacillus cereus was observed. The results indicated that P. ostreatuscultivation is an effective bioconversion process that is capable to transfer agro-wastes into potentially valuable sourceof natural antioxidant and antimicrobial additives for further use in functional food products.