Pleurotus ostreatus mushroom contains important bioactive compounds and has several biological activities; however, mushroom growing substrates have major influence on chemical and functional characteristics of the mushroom. Hence, the study aimed to evaluate the influence of supplementing mushroom growing substrates with wheat bran (WB) towards yield/productivity, bioactive compounds, and antimicrobial and antioxidant activity of P. ostreatus. The mushroom was cultivated on sugarcane substrates supplemented with increasing levels of WB (0%–20%). The mushroom extracts were screened for bioactive compounds using gas chromatography-mass spectrometry (GC-MS). Antimicrobial activity was carried out using microplate assay, while antioxidant potential was investigated using reducing power assay. The addition of supplements on mushroom growing substrates had an influence on mushroom yield; hence, higher supplementation (18% and 20%) produced higher yield. The GC-MS revealed several bioactive compounds with known activity, such as vitamin E, phenol, fatty acids, and terpenoids. Concentration-dependent antioxidant activity was observed; hence, extracts at higher concentrations gave significantly higher reducing power. The P. ostreatus extract had antimicrobial activity against all the tested organisms, with S. aureus showing high susceptibility to most of the extracts. However, mushrooms grown on bagasse substrates supplemented with 14% (0.02 mg/ml) and 20% WB (0.08 mg/ml) proved to have better antimicrobial activity on Escherichia coli. The difference in susceptibility demonstrates that substrates type and composition could have an influence on bioactive compounds found within mushrooms, also influencing medicinal properties of edible mushroom. Thus, supplementing mushroom growing substrates not only improve yield, but also can contribute to bioactive compounds with medicinal potential.