The study demonstrated that cultivation of Pleurotus ostreatus var. florida, Pleurotus eryngii, Pleurotus cornucopiae and Pleurotus djamor on selenium enriched substrate did not significantly affect biological efficiency (%), but total soluble protein content, total phenolic content, flavonoid content and free radical scavenging activity (%) were found to be significantly improved in Se enriched fruit bodies as compared to the control. Elemental analysis of the Se biofortified Pleurotus mushrooms through SEM-EDS showed signals characteristic for selenium on surface of P. ostreatus. var. florida and P. djamor confirming that selenium was incorporated into the cell wall of these fruiting bodies. The Se content was found to be 22.34 μg g−1 dw in Se enriched wheat straw and 0.059 μg g−1 dw in respective non-enriched wheat straw. Se contents of Se-enriched fruit bodies were found to be higher compared to non-enriched Pleurotus spp. FT-IR spectra of proteins from Pleurotus spp. indicated an increase in the flexibility, unfolding, hydrophilicity of the proteins upon Se supplementation.