Toxicity testing on rodents is a prerequisite for using therapeutic agents in humans. However, experiments in mice and rats are expensive and need ethical consideration. Therefore, Galleria mellonella has been extensively used as an alternative model to study microbial infections in humans. The current study focuses on the toxicity of human fungal pathogens, Candida albicans and Aspergillus fumigatus against G. mellonella larvae under controlled laboratory conditions. The injection of both fungal species with different doses (2x10², 2x10³, 2x10⁴, 2x10⁵, and 2x10⁶ cells/larva) gave an LD₅₀ of 6.8x10⁴ and 4.6x10⁴ for A. fumigatus and C. albicans, respectively. Larval, pupal, and adult periods were significantly prolonged at 2x10⁴ for both fungal species and afterward, while pupal and adult developmental ratios and adult longevity were significantly reduced, except at the lowest dose (2x10⁴). Larval treatment with LD₅₀ of both fungal species showed a decrease in the concentrations of proteins, lipids, and carbohydrates in larval tissues, and a greater reduction was observed in larvae injected with C. albicans compared to A. fumigatus. There was a significant decrease in total hemocyte counts (THCs) and hemocyte spreading activity (HSA) after 24 and 48 h of treatment; however, the Variations in plasmatocytes (PLs) and granular cells (GRs) were not statistically significant. In conclusion, this physio-pathological study confirms that changes in some biochemical parameters such as total body proteins, lipids, and carbohydrates as well as cellular immune responses such as changes in hemocyte counts and behavior of Galleria larvae are used as indicators of relative toxicity of the tested pathogens in biomedical research.