The study investigated effect of substrate supplementation with Se alone or in combination with Cu or/and Zn Se on (1) the growth of Pleurotus ostreatus and Pleurotus eryngii; (2) elements accumulation in mushrooms; (3) the antioxidant activities of bio-enriched mushroom extracts in human platelets. The accumulation of elements generally increased over concentration gradient reaching its maximum at 1.2 mM for P. ostreatus and P. eryngii: (1) over 100 and 80 mg kg−1 of Se, respectively (Se supplementation); (2) over 15 and 30 mg kg−1 of Cu, respectively (Se+Cu); (3) over 30 and 85 mg kg−1 of Zn, respectively. Se was predominantly accumulated as an organic fraction. Contrary to P. eryngii, the P. ostreatus biomass decreased with substrate elements concentration but was satisfactory up to 0.9 mM of Se, Se+Cu and Se+Zn. The Se+Cu+Zn model yielded low biomass and elements accumulation. Extracts from mushrooms bio-enriched with Se and Se+Zn (0.6–1.2 mM) revealed significant antioxidant activities in human platelets by ameliorating reactive oxygen species (ROS) and preventing lipid peroxidation. The study demonstrated the potential application of Pleurotus mushrooms as functional food products bio-enriched with essential elements. ROS inhibition by extracts of these mushrooms may be useful in control of platelets activation cascade.