Bio-based alternative leathers may be produced from biomass fiber, protein polymers, bacterial cellulose, and mushroom mycelia. Of these components, mushroom mycelia are of greatest interest. In this study, the potential of Fomes fomentariusas a mushroom mycelial mat was confirmed, and the optimal strain for the development of the mycelial mat was determined. Moreover, the quality of the mycelial mat was improved by identifying an efficient culture method to increase productivity. Mutant strains whose independence was verified were obtained by treatment with gamma irradiation under various conditions. Biofilm formation by the resulting strains was examined in sawdust and liquid media and the characteristics of the biofilms were analyzed. The biofilm of the mutant strains showed results that were similar to or better than the biofilms of longevity and cypress mushrooms. These findings are expected to be utilized in future research aimed at discovering new biomaterials using mushroom mycelia.