Mycelium-based composites (MBCs) are sustainable biomaterials gaining increasing attention, particularly in construction, due to their recyclability, biodegradability, and thermal and acoustic insulation properties. This study investigated the influence of different lignocellulosic biomasses on the mycelial growth of filamentous fungi and their laccase (Lac) production, aiming to identify optimal conditions for MBC development. Seven fungal species—five ascomycetes (Trichoderma longibrachiatum, Talaromyces amestolkiae, Aspergillus flavus, Fusarium oxysporum, Aspergillus niger), one mucoromycete (Rhizopus oryzae), and one basidiomycete (Pycnoporus sanguineus)—were tested for Lac activity, a key enzyme for lignin degradation and biomass colonization. Bamboo, rice husk, and wood sawdust were selected as substrates, previously characterized for bulk density, water absorption, and particle size. Mycelial growth was assessed under two conditions: substrates moistened with distilled water or supplemented with potato dextrose broth (PDB). Fungi with higher Lac activity exhibited greater colonization capacity. PDB supplementation improved growth in species with limited development in water-only conditions. Bamboo showed the highest compatibility among the substrates, likely due to its finer particle size, which may enhance mass transfer and nutrient diffusion. The only strain to produce a cohesive, macroscopically visible composite was P. sanguineus when cultivated on bamboo. These findings highlight the relevance of selecting compatible fungal-biomass pairs and optimizing environmental conditions to advance the development of MBCs as eco-friendly construction materials.