Emergence of resistance among mosquitoes is a recent problem. Safe and eco-friendly agents from biological origins are the need of the hour. Nereistoxin, a naturally occurring substance, was first isolated from the marine annelid Lumbriconereis heteropoda and stored in the freezer. In the present study, the toxicity of nereistoxin was evaluated against vector mosquitoes. The larvicidal, ovicidal and adulticidal activities of nereistoxin were assayed for their toxicity against three important vector mosquitoes, viz., Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). The nereistoxin was inversely proportional to the concentration and directly proportional to the mosquitoes. The larvicidal activity after 24 h LC(50) value was observed at 0.467, 0.535 and 0.601 ppm for A. stephensi, A. aegypti and C. quinquefasciatus, respectively. The ovicidal activity after 120 h zero percentage of egg hatchability was observed at a concentration of 0.8 ppm for A. stephensi and 1.0 ppm for A. aegypti and C. quinquefasciatus. The results of the adulticidal activity after 24 h LD(50) value were observed at 0.022, 0.028 and 0.034 ppm for A. stephensi, A. aegypti and C. quinquefasciatus, respectively. The extracted nereistoxin was characterized and identified by ultraviolet, infrared, nuclear magnetic resonance, mass spectroscopic methods and high pressure liquid chromatography. These results clearly reveal that the nereistoxin served as a potential larvicidal, ovicidal and adulticidal activity against vector mosquitoes.