This work describes the chemical composition of the essential oil from Annona acutiflora and its larvicidal nanoemulsion. The volatile oil was extracted by hydrodistillation. The chemical characterization/quantification was performed by gas chromatography, revealing Î±-santalene, Bicyclogermacrene, and Î±-zingiberene as the major compounds. The nanoemulsion was prepared by low energy method. It was assayed against Aedes aegypti larvae, and in vitro anticholinesterase assay was carried out. This nanoemulsion presented a droplet size around 170 nm and PdI value around 0.170. The assay against larvae showed LC50 and LC90 values after 48 h, respectively, to 21.3 and 37.1 ppm. The anticholinesterase assays showed an IC50 value around 295.6 ppm. These results demonstrate that the method satisfactorily generated fine liquid oily nanodroplets dispersed in aqueous media. Moreover, to our knowledge, this is the first report of the chemical compositionfrom this species, being of great interest for new further integrative practices of vector control.