The present study was conducted to evaluate the mosquitocidal efficiency using a compound isolated from Blumea axillaris (Lam.) DC. against Culex quinquefasciatus and Anopheles stephensi. The compound isolated from leaves of B. axillaris is (4R, 5S) 4-hydroxy-7-angeloyloxycarvatoneacetone. This isolated compound was exposed to eggs and larvae of An. stephensi and Cx. quinquefasciatus and with different concentrations of 0.5, 1.0, 1.5, and 2.0 ppm. Primarily, the hexane extract of an isolated compound exhibited promising larvicidal activities of LC50 and LC90 values of 155.811 and 566.763 ppm against An. stephensi and 77.215 and 99.653 against Cx. quinquefasciatus at 24 hours. Secondly, the compound isolated from B. axillaris at 2-ppm concentration exhibited LC50 and LC90 values of 0.85 and 3.59 and 1.19 and 3.678 ppm against Cx. quinquefasciatus and An. stephensi larvae, respectively. Furthermore, histopathological studies revealed serious damage to the larval midgut cells in both Cx. quinquefasciatus and An. stephensi (treated with compound). Additionally, it showed 81.0% and 84.2% ovicidal activity against both Cx. quinquefasciatus and An. stephensi eggs at 120 h post-treatment. In addition, we also analyzed the influence of isolated compound on the expression of different functional genes of An. stephensi and Cx. quinquefasciatus namely (1) White (Wh) gene, which is responsible for eye pigmentation; (2) the Glutathione transferase (GST) gene that helps for survival when exposed to any insecticides or pesticides; (3) Cytochrome P450 monooxygenase (CYP450) gene that enhances resistance in mosquitoes to digest pesticides and (4) Esterase (Est) gene, which helps in resistance to the variety of insecticides. These overall studies and results clearly suggest that compounds from B. axillaris could act as good mosquitocidal agents against both Cx. quinquefasciatus and An. stephensi and the compound were also first time reported as mosquitocidal activity.