Mitonuclear interactions are crucial in governing mitochondrial function, development and responses to stress in eukaryotic organisms. In this study, we explored how varying mitochondrial haplotypes affect the phenotype and oxidative stress response using hybrids of the basidiomycete Pleurotus ostreatus (P. ostreatus) as a model system. By performing reciprocal crosses between monokaryotic strains with distinct nuclear and mitochondrial genomes, we identified notable differences in growth rates, accumulation of reactive oxygen species (ROS) and gene expression patterns. Hybrids with incompatible mitonuclear combinations displayed slower growth and elevated expression of genes — some showing transgressive inheritance — associated with the Electron Transport Chain (ETC) and antioxidant defences. Mitochondria-dependent heterosis was observed in hybrids sharing the same nuclear background, but differing in mitochondrial genome, suggesting that mitonuclear incompatibilities can result in oxidative imbalance and compromised fungal performance. This experimental approach opens wide possibilities for exploring mitonuclear interactions and highlights the significance of mitonuclear co-adaptation in an edible mushroom, offering valuable insights for enhancing hybrid breeding programmes by accounting for the role of mitonuclear interactions in shaping quantitative traits related to mushroom yield.