This study investigates the insecticidal potential of Aspergillus melleus protease on Galleria mellonella larvae, revealing significant biological effects, including increased mortality, developmental delays, adverse impacts on nutritional indices, and histopathological changes. Fungal spores were isolated from soil in El-Khatatba and cultured to produce protease enzyme, with optimal production occurring at pH 8, 35°C, and 72 hours of incubation.  A dose-dependent increase in larval and pupal mortality was observed, with LC₅₀ and LC₉₀ values determined as 1.9 × 10⁶ and 2.5 × 10⁸ spores/mL, respectively. Spore concentrations significantly delayed the larval and pupal development and induced morphological deformities in G. mellonella. Histopathological examination revealed damage to the midgut epithelial layer of the larvae, contributing to impaired nutrient absorption. Nutritional indices revealed a marked decrease in relative growth and consumption rates, as well as reduced conversion efficiency and increased feeding deterrence. These findings highlight the potential of A. melleus protease as a bio-insecticide, with G. mellonella serving as a model insect for evaluating its effectiveness.