Pleurotus giganteus, a heat-tolerant mushroom with high nutritional and medicinal value, is a promising species for tropical mushroom cultivation in Hainan, China. However, its current dependence on rubber sawdust as the primary substrate compromises environmental sustainability. In this study, we applied a “replacing wood with grass” strategy and used a simplex-lattice design to optimize substrate formulations based on agro-residues. Laboratory screening identified banana straw and chili straw as effective substitutes for rubber sawdust, supporting rapid and dense mycelial growth. Mixed formulations showed distinct advantages in mycelial growth, enzyme activity, agronomic traits (growth cycle, yield, and cap-to-stipe ratio), and nutritional composition compared to the control formulation (CF), particularly in terms of growth rate and laccase activity. Correlation analyses revealed that both individual ingredients and their interactions significantly affected mycelial growth and agronomic traits, with the magnitude and direction of effects depending on their relative proportions. Based on expected response values for key evaluation indices, an optimal formulation (9.97% rubber sawdust, 24.33% banana straw, 10.70% chili straw, 40% cottonseed hulls, 10% wheat bran, and 5% lime) was predicted and experimentally validated to outperform the CF. This study provides a sustainable basis for localized cultivation of P. giganteus in Hainan and supports the high-value valorization of agricultural residues for mushroom production.

Hericium erinaceus, a rare edible–medicinal fungus, has attracted great attention in food and pharmaceutical fields due to its rich nutritional and bioactive components. However, its traditional cultivation relies heavily on wood chip substrates, causing resource unsustainability. The “wood-replacing-with-grass” technology can address this issue, contributing to ecological conservation and alleviating resource conflicts between edible fungus cultivation and forestry development. This study focused on straw substitution for wood chips, initially screening suitable straw types and optimal addition ratios from 7 straw varieties, and systematically investigating the agronomic trait variations in H. erinaceus under different substrate formulations via cultivation experiments. Results showed the following: (1) Rapeseed straw, soybean straw, and corn straw substituting 20%, 30%, and 40% of wood chips, respectively, promoted better mycelial growth of H. erinaceus. (2) All screened straw formulations enabled fruiting. With increased straw addition, the mycelial full colonization time shortened (up to 5 days shorter in 40% corn/soybean straw treatments). The 20% corn straw treatment showed significantly higher biological efficiency and average fresh weight than the control (CK); the 20% soybean straw treatment had no significant difference in biological efficiency but significantly higher average fresh weight than CK; and the 20% rapeseed straw treatment showed no significant differences in both indexes from CK. However, when straw addition exceeded 20%, fruiting body firmness, yield, and biological efficiency decreased progressively. (3) The 40% soybean straw treatment yielded fruiting bodies with the highest crude protein, manganese, and iron contents, while the 40% rapeseed straw treatment had the highest crude fat, potassium, phosphorus, calcium, zinc, and selenium contents. These findings provide a theoretical basis and practical reference for optimizing H. erinaceus cultivation substrate formulations, improving product quality, and promoting sustainable industrial development.