Abstract <p>Mycelium-based materials derived from white-rot fungi have attracted increasing attention as sustainable and eco-friendly alternatives to conventional products. However, their mechanical strength and durability remain relatively inferior. Molecular breeding of white-rot fungi offers a promising strategy to address these limitations. Recent studies have suggested that mycelial density and cell wall structure play key roles in determining the physical properties of mycelium-based materials. In this study, we disrupted <i>mbp1</i>, which encodes a transcription factor required for normal mycelial growth and cell wall synthesis, in the white-rot fungus <i>Pleurotus ostreatus</i> and investigated the effects on mycelium mats and mycelium-based composites. The mycelium mats of the dikaryotic <i>mbp1</i> disruptants exhibited higher Young’s moduli and ultimate tensile strengths than those of the control strain (20b×#61), indicating that <i>mbp1</i> disruption resulted in stiffer mycelium mats. This may be because of the increased mycelial density in the dikaryotic <i>mbp1</i> disruptants. In addition, the dikaryotic <i>mbp1</i> disruptants produced harder mycelium-based composites than the strain 20b×#61. These findings indicate that <i>mbp1</i> disruption improved the characteristics of mycelium-based composites. To our knowledge, this is the first study demonstrating that molecular breeding can enhance the performance of mycelium-based composites, thereby paving the way for the development of efficient strategies to advance mycelium-based materials.</p> Key points <p>• <i>mbp1 disruption resulted in stiffer mycelium mats.</i></p> <p>• <i>Dikaryotic mbp1 disruptants formed thinner but denser mycelium mats.</i></p> <p>• <i>mbp1 disruption improved the mechanical strength of mycelium-based composites.</i></p>