Fungal mycelium, renowned for its robust fiber structure, is gaining widespread attention as a sustainable alternative to traditional plastics and textiles. Strain optimization offers the opportunity to improve these mycelial materials by systematically selecting specific phenotypes that have ideal mechanical and physiochemical properties. Schizophyllum commune, the common split gill mushroom, is a cosmopolitan species with over 23 000 mating types and abundant genetic diversity. In this study, this species was used as a model to explore the potential of leveraging natural genetic variation within species to develop fungal mycelial materials with diverse properties. Specifically, four divergent monokaryotic strains of S. commune sourced globally were selected, and through mating, 12 dikaryotic progeny, each with their unique combinations of nuclear and mitochondrial deoxyribonucleic acid (DNA) were derived. These 16 strains were assessed for their growth in both solid and liquid media. Their mycelia from liquid media were further processed, including by linking with two different crosslinkers, polyethylene glycol 400, and glycerol, to form mycelial films. Mechanical testing and surface characterization showed that the mycelial films differed greatly in a diversity of features, from water retention to strength, ductility, morphology, and hydrophobicity. Moreover, Fourier transform infrared spectroscopy showed that different strains had unique chemical fingerprints revealing diverse cell wall composition that interfaced with each of the crosslinkers uniquely. Statistical analyses revealed that, along with the highly influential crosslinker effects, nuclear-mitochondrial genotype interactions were key factors in tuning the performances of these materials. The two-layer tunability of the fungal materials points to the novel potential for genetically optimized strains, such as through protoplasting to separate nuclei in dikaryons to monokaryons with new nuclear-mitochondrial combinations and/or protoplast fusion to artificially create novel dikaryons, with tailored mycelial materials properties for applications in textiles, coatings, and mycoremediation.