Background: Nanoemulsion composed of neem oil and non-ionic surfactant Tween 20, with a mean droplet size ranging from 31.03 to 251.43 nm, was formulated for various concentrations of the oil and surfactant. The larvicidal effect of the formulated neem oil nanoemulsion was checked against Culex quinquefasciatus. Results: O/W emulsion was prepared using neem oil, Tween 20 and water. Nanoemulsion of 31.03 nm size was obtained at a 1:3 ratio of oil and surfactant, and it was found to be stable. The larger droplet size (251.43 nm) shifted to a smaller size of 31.03 nm with increase in the concentration of Tween 20. The viscosity of the nanoemulsion increased with increasing concentration of Tween 20. The lethal concentration (LC 50) of the nanoemulsion against Cx. quinquefasciatus was checked for 1:0.30, 1:1.5 and 1:3 ratios of oil and surfactant respectively. The LC 50 decreased with droplet size. The LC 50 for the ratio 1:3 nanoemulsions was 11.75 mg L -1. Conclusion: The formulated nanoemulsion of 31.03 nm size was found to be an effective larvicidal agent. This is the first time that a neem oil nanoemulsion of this droplet size has been reported. It may be a good choice as a potent and selective larvicide for Cx. quinquefasciatus. © 2011 Society of Chemical Industry.