Mycelium-based leather is a promising sustainable material that offers a biodegradable and animal-free alternative to conventional leather. However, ensuring its consistent quality during mass production remains a significant challenge owing to variability in biological growth and vulnerability to contamination. This study investigated the effects of surface modification and plasticization treatments on the structural and chemical properties of mycelial mats. Structural and chemical profile alterations were evaluated using hyperspectral imaging (HSI), Fourier-transform infrared spectroscopy, and scanning electron microscopy. HSI effectively detected these changes and surface defects nondestructively. It enabled precise differentiation between treated and untreated regions, thus supporting rapid and spatially resolved quality inspection. These results highlight the potential of HSI as an effective tool for evaluating the effects of pretreatment and monitoring contamination in mycelium-based leather production.