Mosquitoes are vectors for numerous public health–threatening diseases. This study investigates an eco-friendly bioinsecticidal compound, (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl) benzoic acid, isolated from Bacillus pumilus, against Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi. The compound was characterized using TLC, UV–Vis spectroscopy, FTIR, NMR, and GC–MS. Dose-dependent mortality rates reached up to 100% in target species, with LC50 values of 13.65, 14.90, and 9.64 ppm, respectively. The compound enhanced antioxidant enzyme levels, caused DNA damage, and induced histopathological larval changes, while remaining safe for Gambusia affinis. This study demonstrates a species-specific, environmentally friendly alternative to synthetic insecticides for mosquito control. © 2022 Elsevier B.V., All rights reserved.