The composition of the substrate is one of the most critical factors influencing the quality as well as the nutritional value and bioactive content of mushrooms. Therefore, the effects of various substrates, such as barley and oat straw (BOS), beech wood shavings (BWS), coffee residue (CR), rice bark (RB) and wheat straw (WS, control substrate), on the biochemical properties (lipid, protein, polysaccharide, glucan, ash, and mineral content, fatty acids and tocopherols composition), total phenolic compounds and antioxidant activity of Pleurotus mushrooms, P. ostreatus (strains AMRL 144, 150) and P. eryngii (strains AMRL 166, 173-6), cultivated in ‘bag-logs’, was examined. Proximate analysis of A. bisporus and A. subrufescens grown on two different composts (C/N ratios of 10 and 13) was conducted, too. The whole carposomes, pilei and stipes were analyzed. Results showed that BOS, RB, BWSs and CR improved the antioxidant activity of Pleurotus species and their nutritional characteristics. Both pilei and stipes were rich in polysaccharides (27.51–67.37 and 22.46–39.08%, w/w, for Pleurotus and Agaricus spp., respectively), lipids (0.74–8.70 and 5.80–9.92%, w/w), proteins (6.52–37.04 and 25.40–44.26, w/w, for Pleurotus and Agaricus spp., respectively) and total phenolic compounds (10.41–70.67 and 7.85–16.89 mg gallic acid equivalent/g for Pleurotus and Agaricus spp., respectively), while they contained important quantities of unsaturated FAs of nutritional and medicinal importance. Pilei were richer in proteins, total phenolic compounds and enhanced antioxidant activity and reducing power than stipes, whereas stipes were richer in IPSs and glucans compared to the corresponding pilei. Thus, mushroom cultivation could upgrade rejected agro-industrial residues and wastes to new uses as substrates for the production of mushrooms with specific nutritional and medicinal attributes.