This study evaluated the antifungal activity of four fruiting bodies of oyster mushroom harvested from three agro-substrates in vitro. At three concentrations (2, 4 and 8 mg/disc), extracts discs of Pleurotus ostreatus (grey), P. ostreatus var. florida, P. cornucopiae var. citrinopileatus and P. salmoneostramineus were tested against three fungal pathogens: Trichoderma harzianum (after 2 days), Verticillium sp. and Pythium sp. (after 5 days) via the Disc Diffusion Method. The highest overall activity was by the extract disc Y2 (P. cornucopiae grown on M2 substrate; 70% wheat straw, Owaid; M.N.; Al-Saeedi, S.S.S.; Al-Assaffii, I.A.A.Antifungal activity of cultivated oyster mushrooms on various agro-wastes. Summa Phytopathologica, v.43, n.1, p.09-13, 2017.20% hardwood sawdust and 10% date palm fibers) and the lowest by Y1 (P. cornucopiae grown on wheat straw). The best inhibition zone was 16 mm toward T. harzianum by extract disc W2 (2 mg/disc) (P. ostreatus var. florida grown on M2 substrate), compared with 23 mm with Nystatin disc (100 U), followed 7 and 5 mm by P3 (P. salmoneostramineus grown on M3 substrate; 50% wheat straw, 30% hardwood sawdust and 20% date palm fibers) extract disc (8 mg/disc) against Pythium sp., and (4 mg/disc) against Verticillium sp., respectively.Keywords: Pleurotus spp., agro-wastes, contaminated fungi, antifungal activity, bioactivity.The oyster mushroom, Pleurotus spp., is edible and belongs to the fungi kingdom, phylum Basidiomycota (32). About seventy species of Pleurotus spp. have been recorded and new species are being discovered. Many oyster mushrooms are primary decomposers of hardwood trees found worldwide (14). Thus, it can be cultivated on a wide variety of substrates containing lignin, cellulose and hemicellulose (26). It must obtain nutrients from such organic sources as dead organisms since they had absorbed nutrients after digesting large molecules into smaller units because of their secreted enzymes (10); thus, it has been grown in Iraq on various agro-wastes in the wild (18), or manually (5, 17) on cardboard (19,20), date palm wastes (21), and tree sawdust (24).Since ancient times, macrofungi have been used as a valuable food source and as traditional medicines around the world. The fungi constitute an important source for some compounds including enzymes and antibiotics (9). Consequently, the antimicrobial activity of various polysaccharides from medicinal mushrooms is being reevaluated in relation to their clinical efficacy, given that such compounds would be expected to function to ward off bacterial and fungal infections resistant to current antibiotics (31). Medicinal mushrooms are able to synthesize a great amount of secondary metabolites that present antitumoral, antiviral, anti-inflammatory (8), antibacterial, antifungal (23) and anti-yeast activities (25).Currently, a large range of mushrooms species are grown in liquid media. The obtained mycelia used for various applications, such as obtaining dietary supplements, pharmaceutical applications, convers...