Diseases transmitted by vectors such as Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) have been responsible for endemics and epidemics in several countries, causing irreparable damage to human health. For this reason, vector control is one of the main strategies to prevent the contamination and dissemination of these etiological agents. In this study, the essential oil (EO) of Ocimum basilicum was obtained by hydrodistillation, and the compounds were identified by GC/MS. A nanoemulsion was obtained through a low-energy input method and evaluated by photon correlation spectroscopy. Bioassays were performed against 4th instar larvae of A. aegypti and C. quinquefasciatus. Furthermore, additional in silico studies of biological activity prediction and molecular docking for the acetylcholinesterase enzyme and juvenile hormone protein targets were performed with the 53 identified compounds of the EO. The major compounds identified in greater quantity were linalool (32.66%) and anethole (32.48%). The obtained nanoemulsion had an average size diameter between 244.6 and 280.4 nm and a polydispersity index below 0.250 during the entire storage period. The nanoemulsion was tested at concentrations of 10, 20, 30, 40, and 50 mg/L and the following activity values were observed: LC₅₀ = 42.15 mg/L and 40.94 mg/L and LC₉₀ = 50.35 mg/L and 48.87 mg/L for A. aegypti (24 h and 48 h); LC₅₀ = 39.64 mg/L and 38.08 mg/L and LC₉₀ = 52.58 mg/L and 54.26 mg/L for C. quinquefasciatus (24 h and 48 h). The in silico results showed better activity values for linalool, anethole, carvone, α-selinene, eugenol, and limonene. The α-selinene compound showed the best binding affinity with the insect acetylcholinesterase enzyme (−9.1 Kcal) in molecular docking, showing the importance of antagonist compounds in elucidating the mechanism of action for the investigated targets. Thus, the studied nanoemulsion was considered active against the tested species, becoming a potential alternative as an ecological bioinsecticide due to bioactivity and simplicity of formulation.