Annona mucosa Jacq. (Annonaceae) is an edible plant whose fruit is appreciated in the Amazon region of Brazil. The seeds of this plant are rich sources of acetogenins, substances that have antimicrobial, anticancer, repellent, insecticide and larvicidal activities. However, studies on larvicidal evaluation of these substances are almost always limited to the species Aedes aegypti, with few studies with the species Aedes albopictus. In this research, it was investigated the larvicidal effect and mechanisms of action of the ethanolic extract and of the main constituents of Annona mucosa against Ae. aegypti and Ae. albopictus. The compounds were isolated from the ethanolic seed extract by successive silica gel chromatographic columns and characterized by NMR and Infrared spectroscopic analysis, as well as by comparison with literature data, as the acetogenins rolliniastatin 1 and rollinicin. Third- and fourth-instar larvae were submitted to different concentrations of ethanolic extract, rolliniastatin 1 and rollinicin to estimate the LC₅₀ and LC₉₀ after 24 h exposure. The ethanolic extract and rolliniastatin 1 were more active against Ae. albopictus, with LC₅₀ values of 0.55 and 0.20 μg.mL⁻¹, respectively, compared with Ae. aegypti, on which they exhibited values of 2.60 and 0.43 μg.mL⁻¹, respectively. Rollinicin was, on the contrary, more active against Ae. aegypti (LC₅₀ of 0.78 μg.mL⁻¹) than against Ae. albopictus (LC₅₀ of 1.13 μg.mL⁻¹). Significant differences were observed among enzymatic activities of larvae treated with natural products obtained from Annona mucosa and histopathological analysis of the midgut of treated larvae suggested the existence of two action mechanisms. EEAM and main acetogenins, rolliniastatin 1 and rollinicin did not present toxicity against zebrafish. The results demonstrate the larvicidal potential of seeds ethanolic extract and acetogenins from A. mucosa as green larvicidal agents for the control of Ae. aegypti and Ae. albopictus.