Essential oils are important natural resource of pesticides/insecticides. Thus its can to represent an alternative source of chemicals to control disease vectors. The essential oils from Alpinia speciosa, Cymbopogon citratus and Rosmarinus officinalis are very active against Aedes aegypti killing one-hundred percent of the larvae in a few minutes. The mosquito larvae V-ATPases have great importance, because they are responsible to energize the process of alkalinization in the anterior region of the midgut lumen, sustaining large longitudinal pH gradient. Moreover, these enzymes are also involved in the transepithelial transport removing electrolytes and fluids from Malpighian tubules. The objective of this study was to evaluate the effect “in vivo” of the essential oils A. speciosa, C. citratus and R. officinalis against V-type H⁺-ATPase from Ae. aegypti larvae in order to study their mode of action. The larvicidal properties of the essential oils were tested and the DL₅₀ were calculated.  All essential oils caused 100 % of mortality with DL₅₀ of 1.06 mL/mL, 0.62 mL/mL and 1.87 mL/mL to A. speciosa, C. citratus and R. officinalis, respectively. Larval V-ATPases preparations were obtained by differential centrifugation. Total and ATPase NO₃^-sensitive activities were studied by determining inorganic phosphate liberated from the substrate ATP. Thirty minutes of exposure to the essential oils (0.4 mL/ mL) affected ATPases activities revealing inhibition of the total ATPases in 45.73 % (A. speciosa); 49.24 % (C. citratus); 31.49 % (R. officinalis). The specific activities V-ATPases (NO₃^- -sensitive) were inhibited in 49.95 % (A. speciosa); 52.12 % (C. citratus); 40.16% (R. officinalis).These results points the Ae.aegypti larvae V-ATPases as a potential target of the toxic effect of essential oils of the A. speciosa, C. citratus and R. officinalis.