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The present research investigated the chemical characterization and insecticidal activity of <i>n</i>-Hexane extracts of <i>Epaltes divaricata</i> (NH-EDx) along with their chief derivatives <i>n</i>-Hexadecanoic acid (<i>n</i>-HDa) and <i>n</i>-Octadecanoic acid (<i>n</i>-ODa) against the dengue vector <i>Aedes aegypti</i> and lepidopteran pest <i>Spodoptera litura</i>. Chemical screening of NH-EDx through GC–MS analysis delivered nine major derivatives, and the maximum peak area percentage was observed in <i>n</i>-Hexadecanoic acid (14.63%) followed by <i>n</i>-Octadecadienoic acid (6.73%). The larvicidal activity of NH-EDx (1000 ppm), <i>n</i>-HDa (5 ppm), and <i>n</i>-ODa (5 ppm) against the <i>A. aegypti</i> and <i>S. litura</i> larvae showed significant mortality rate in a dose-dependent way across all the instars. The larvicidal activity was profound in the <i>A. aegypti</i> as compared to the <i>S. litura</i> across all the larval instars. The sublethal dosages of NH-EDx (500 ppm), <i>n</i>-HDa (2.5 ppm), and <i>n</i>-ODa (2.5 ppm) also showed alterations in the larval/pupal durations and adult longevity in both the insect pests. The enzyme activity revealed that the α- and β-carboxylesterase levels were decreased significantly in both the insect pests, whereas the levels of GST and CYP450 uplifted in a dose-dependent manner of NH-EDx, <i>n</i>-HDa, and <i>n</i>-ODa. Correspondingly, midgut tissues such as the epithelial layer (EL), gut lumen (GL), peritrophic matrix (Pm), and brush border membrane (BBM) were significantly altered in their morphology across both <i>A. aegypti</i> and <i>S. litura</i> against the NH-EDx and their bioactive metabolites. NH-EDx and their bioactive metabolites <i>n</i>-HDa and <i>n</i>-ODa showed significant larvicidal, growth retardant, enzyme inhibition, and midgut toxicity effects against two crucial agriculturally and medically challenging insect pest of ecological importance.