The necessity for environmentally safe choices for synthetic larvicides rises day by day due to severe global mosquito-borne ailments. In this concern, the larvicidal activity was conducted with the ethanolic leaf extract of Cleome gynandra against Aedes aegypti by bioassay and molecular mechanistic approaches. The phytochemicals present in the Cleome gynandra were identified by GC-MS techniques, and palmitic acid and linolelaidic acid were identified as the major and more bioactive constituents among all. Larvicidal bioanalysis explored a clear dose-dependent mortality with significant larval death, and the LC₅₀ value was found to be 15.39 µg/mL indicates effective larval inhibition even at low doses. To validate this bioassay, a molecular docking study was performed with 4EY7 protein, and the interactions of palmitic acid and linolelaidic acid with binding energies of -7.0 kcal/mol and -7.8 kcal/mol corroborate this effective larval control. Conclusively, all the findings showed that the phytoconstituents present in the ethanolic leaf extract of Cleome gynandra act as an effective, eco-friendly, and sustainable mosquito vector control agent.