Abstract Nanotechnology has emerged as a promising area for innovative products, including insecticides. Dengue is a tropical disease which is considered a critical health problem in developing countries, due to negative impacts to the environment caused by synthetic chemicals used for vector control (Aedes aegypti). Thus, developing of natural products based insecticidal are considered very promising. On this context, the aim of the present study was to obtain an O/W nanoemulsion containing Rosmarinus officinalis L., Lamiaceae, essential oil and evaluate its larvicidal activity against A. aegypti. Low energy method was employed, allowing achievement of small droplets. The nanoemulsion also presented low polydispersity and mean droplet below 200 nm, even after 30 days of storage. Potential mortality levels were observed after 24 h (80 ± 10%) and 48 h (90 ± 10%) in A. aegypti larvae at final concentration of 250 ppm, related to R. officinalis essential oil. This study contributes to nanobiotechnology of natural products, presenting a potential larvicidal nanoemulsion prepared with R. officinalis essential oil. Moreover, nanoemulsion production involved a non-heating procedure, describing easy technique which may be useful for integrative control programs.