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Background Chromolaena odorata , has been traditionally known for its insect repellent property. Aim of this study was to determine larvicidal tendency of C. odorata on Culex quinquefasciatus and isolate compounds responsible for this activity and to determine the mechanism of action of these compounds. Methods C. odorata plant extract was screened for mosquito larvicidal activity. The extract was fractionated using chromatography and the bioactive fraction showing larvicidal activity was identified. The chemical nature of the compounds in the bioactive fraction was determined using NMR and Mass spectrometry. Results We identified phytosterols and alkanols to be the compounds regulating larvicidal activity in the bioactive fraction of the plant extract. Stigmasterol and 1-hexacosanol were identified to be the chief orchestrators of larvicidal activity and their mode of action has been observed to be neurotoxicity. At a molecular level both stigmasterol and 1-hexacosanol were found to be inhibiting acetylcholinesterase activity in C. quinquefasciatus & A. aegypti. The acetylcholinesterase inhibitory effect was validated in vitro using recombinant acetylcholinesterase and ex vivo in larval homogenates of Culex and Aedes . Electrophysiological studies using electroantennography have shown enhanced neural response to these compounds. Conclusions Neurotoxic effect of C. odorata derived stigmasterol and 1-hexacosanol, exerted through acetylcholinesterase inhibition was responsible for the mortality of C. quinquefasciatus , A. aegypti & Chironomus riparius . EAG studies pointed out hyper-excitability of the olfactory system by these compounds. General significance These compounds are natural agents for mosquito control that can be used in vector control as larvicidal compounds, pending further investigations.