The increasing risk of vector-borne diseases and the environmental pollution in the day-to-day life due to the usage of the conventional pesticides makes the role of nanotechnology to come into the action. The current study deals with one of the applications of nanotechnology through the formulation of neem urea nanoemulsion (NUNE). NUNE was formulated using neem oil, Tween 20, and urea using the microfluidization method. Prior to the development of nanoemulsion, the ratio of oil/surfactant/urea was optimized using the response surface modeling method. The mean droplet size of the nanoemulsion was found to be 19.3 ± 1.34 nm. The nanoemulsion was found to be stable for the period of 4 days in the field conditions which aids to its mosquitocidal activity. The nanoemulsion exhibited a potent ovicidal and larvicidal activity against A. aegypti and C. tritaeniorhynchus vectors. This result was corroborated with the histopathological analysis of the NUNE-treated larvae. Further, the effect of NUNE on the biochemical profile of the target host was assessed and was found to be efficacious compared to the bulk counterpart. The nanoemulsion was then checked for its biosafety towards the non-target species like plant beneficial bacterium (E. ludwigii), and phytotoxicity was assessed towards the paddy plant (O. sativa). Nanometric emulsion at the concentration used for the mosquitocidal application was found to be potentially safe towards the environment. Therefore, the nanometric neem-laced urea emulsion tends to be an efficient mosquito control agent with an environmentally benign property.