BackgroundAedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit dengue fever and West Nile virus diseases, respectively. This study was conducted to determine the toxicity and mechanism of action of four flavonoids and two fatty acids from Millettia pinnata (Fabaceae) seed as well as six pure fatty acids and four fatty acid esters toward third instar larvae from insecticide-susceptible C. pipiens pallens and A. aegypti as well as wild A. albopictus. Efficacy of 12 experimental liquid formulations containing M. pinnata seed methanol extract and hydrodistillate (0.5–10.0% liquids) was also assessed.MethodsThe contact toxicities of all compounds and 12 formulations were compared with those of two larvicides, temephos and fenthion and the commercial temephos 200 g/L emulsifiable concentrate (EC). The possible mode of larvicidal action of the constituents was elucidated using biochemical methods. Larval mortality and cAMP level were analyzed by the Bonferroni multiple-comparison method.ResultsPotent toxicity was produced by karanjin, oleic acid, karanjachromene, linoleic acid, linolenic acid, pongamol, pongarotene, and elaidic acid toward C. pipiens pallens larvae (24 h LC50, 14.61–28.22 mg/L) and A. aegypti larvae (16.13–37.61 mg/L). Against wild A. albopictus larvae, oleic acid (LC50, 18.79 mg/L) and karanjin (35.26 mg/L) exhibited potent toxicity. All constituents were less toxic than either temephos or fenthion. Structure–activity relationship indicates that the degree of saturation, the side chain length, and the geometric isomerism of fatty acids appear to play a role in determining the fatty acid toxicity. Acetylcholinesterase (AChE) is the main site of action of the flavonoids, oleic acid, and palmitic acid. The mechanism of larvicidal action of elaidic acid, arachidic acid, and behenic acid might be due to interference with the octopaminergic system. Linoleic acid and linolenic acid might act on both AChE and octopaminergic receptor. M. pinnata seed extract or hydrodistillate applied as 10% liquid provided 100% mortality toward the three mosquito species larvae and the efficacy of the liquids was comparable to that of temephos 200 g/L EC.ConclusionFurther studies will warrant possible applications of M. pinnata seed-derived products as potential larvicides for the control of mosquito populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0848-8) contains supplementary material, which is available to authorized users.