Aedes 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 seven fatty acids from Millettia pinnata (Fabaceae) seed as well as four fatty acid esters toward third instar larvae from insecticide-susceptible C. p. pallens and A. aegypti as well as wild A. albopictus. Potent toxicity was produced by karanjin, oleic acid, karanjachromene, linoleic acid, linolenic acid, pongamol, pongarotene, and elaidic acid toward C. p. 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. Acetylcholinesterase (AChE) is the main site of action of the flavonoids, oleic acid, and palmitic acid.,Linoleic acid and linolenic acid might act on both AChE and octopaminergic receptor. Further studies will warrant possible applications of M. pinnata seed-derived products as potential larvicides for the control of mosquito populations.