Entomopathogenic fungi are a promising category of biocontrol agents with mosquitocidal properties. Prior studies have proved their potential to reduce fecundity, human biting and vector competence, all of them together determine vectorial capacity of the mosquitoes. Unfortunately, conventional vector control strategies are inadequate with growing problem of insecticide resistance and environmental deterioration. Therefore, alternate vector control measures are immediately needed and to accomplish that, an improved understanding of behavioral and physiological defense mechanisms of the mosquitoes against fungal infection is essential. In this study, fitness was considered with respect to different behavioral (self-grooming and flight), physiological (antifungal activity and antimicrobial peptides) parameters and survival rates as compared to the control group. We found a significant upregulation in CLSP2, TEP22, Rel1 and Rel2 genes at multiple time periods of fungal infection, which indicates the successful fungal infection and activation of Toll and IMD pathways in mosquitoes. RNAi-mediated silencing of Rel1 and Rel2 genes (transcription factors of Toll and IMD pathways, respectively) significantly reduced the survival, self-grooming frequencies and durations, and flight locomotor activity among adult Ae. aegypti female mosquitoes. Moreover, Rel1 and Rel2 knockdown significantly decreased antifungal activity and antimicrobial peptides expression levels in target mosquitoes. These results indicate an overall decrease in fitness of the mosquitoes after fungal challenge following Rel1 and Rel2 silencing. These findings provide an improved understanding of behavioral and physiological responses in mosquitoes with altered immunity against entomopathogenic fungal infections which can guide us towards the development of novel biocontrol strategies against mosquitoes.