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The present investigation aimed to determine the fungal toxicity of <i>Isaria tenuipes</i> (My-It) against the dengue mosquito vector <i>Aedes aegypti</i> L. and its non-target impact against the aquatic predator <i>Toxorhynchites</i><i>splendens</i>. Lethal concentrations (LC₅₀ and LC₉₀) of My-It were observed in 2.27 and 2.93 log ppm dosages, respectively. The sub-lethal dosage (My-It-1 × 10⁴ conidia/mL) displayed a significant oviposition deterrence index and also blocked the fecundity rate of dengue mosquitos in a dose-dependent manner. The level of major detoxifying enzymes, such as carboxylesterase (α-and β-) and SOD, significantly declined in both third and fourth instar larvae at the maximum dosage of My-It 1 × 10⁵ conidia/mL. However, the level of glutathione S-transferase (GST) and cytochrome P-450 (CYP450) declined steadily when the sub-lethal dosage was increased and attained maximum reduction in the enzyme level at the dosage of My-It (1 × 10⁵ conidia/mL). Correspondingly, the gut-histology and photomicrography results made evident that My-It (1 × 10⁵ conidia/mL) heavily damaged the internal gut cells and external physiology of the dengue larvae compared to the control. Moreover, the non-target toxicity against the beneficial predator revealed that My-It at the maximum dosage (1 × 10²⁰ conidia/mL) was found to be less toxic with <45% larval toxicity against <i>Tx.</i><i>splendens</i>. Thus, the present toxicological research on <i>Isaria tenuipes</i> showed that it is target-specific and a potential agent for managing medically threatening arthropods.