Pleurotus mushrooms are valuable food supplements with health and environmental restorative potential. In this paper, we sought to evaluate the biological activities and profile the bioactive compounds found in Pleurotus ostreatus cultivated from agro-waste supplemented with maize flour. We investigated carbon to nitrogen (C/N), antimicrobial, antioxidant, and antimalarial potential for the varying supplementation during mushroom cultivation. GCMS was utilized for screening bioactive compounds found in P. ostreatus. Changes in supplementation directly correlate with changes in compound profiling. Nonetheless, some compounds were found to be common amongst the tested mushrooms, including pentadecanoic acid; 9,12-octadecadienoic acid, methyl ester; pentadecanoic acid, methyl ester; octadecanoic acid; and diisooctyl phthalate. The highest antimicrobial potential against Gram-positive Staphylococcus aureus was observed when maize flour supplements were increased to 12% and 18%. Our data demonstrated that the observed antioxidant (DPPH, ABTS, and reducing power) and antimicrobial activity could emanate from various supplementation conditions. Furthermore, supplementation has an impact on the mushroom yield and phytochemical profiles of the produced mushroom.