The mosquitoes vectors of the genus Aedes and Anopheles present resistance to several commercial insecticides, which are also toxic to non-predators targets. On the other hand, essential oils are a promising source of insecticides. Thus, in this work, the essential oil from the leaves of Piper purusanum was characterized by gas chromatography based approaches and evaluated as biodefensive against malaria and dengue vectors. The main compounds of P. purusanum essential oil were β-caryophyllene (57.05%), α-humulene (14.50%) and germacrene D (8.20%). The essential oil inhibited egg hatching (7.6 ± 1.5 to 95.6 ± 4.5%) caused larval death (LC 50 from 49.84 to 51.60 ppm) and inhibited the action of acetylcholinesterase (IC 50 of 2.29 µg/mL), which can be related to the mechanisms of action. On the other hand, the biological activity of β-caryophyllene, α-humulene and germacrene D were higher than the essential oil. In addition, these sesquiterpenes and essential oil did not show a lethal effect on Toxorhynchites splendens, Anisops bouvieri, Gambusia a nis and Diplonychus indicus (LC 50 from 2098.80 to 7707.13 ppm), although D. indicus is more sensible (SI/PSF from 48.56 to 252.02 ppm) to essential oil, representing a natural alternative against these relevant vectors.* Con dence limit of 95%. Letters in the same column indicate statistical differences (ANOVA and Tukey p < 0.05). r 2 -Linear regression.