The effectiveness of lime essential oil (EO) from Citrus aurantifolia as a larvicide was investigated on third-instar larvae of Culex pipiens mosquitoes, with an LC50 value of 35.4ppm. The larvae exhibited morphological aberrations, including hardened, darkened, and shrunken anal papillae, as well as darkening of the siphon. After treatment, there was a reduction in total body protein (15.3 ± 0.21), along with an increase in the levels of acid phosphatase and alkaline phosphatase (4.67 ± 0.12 and 36.03 ± 0.79, respectively). Conversely, the activity of acetylcholinesterase and glutathione S-transferase decreased (13.07 ± 0.27 and 312.67 ± 7.22, respectively). The treated larvae also showed significant histopathological changes in the cuticle and midgut tissues. These findings indicate that a nanoemulsion of lime EO from Citrus aurantifolia is a promising natural larvicide that is biodegradable, environmentally friendly, and effective for controlling Culex pipiens, a vector of disease.