The present study aimed to analyze the chemical composition of the essential oil from Garcinia gardneriana (Planchon & Triana) Zappi leaves and fruits, and to determine its acaricidal activity on Rhipicephalus microplus by larval packet test and larvicidal action on Aedes aegypti by larval immersion test. The chemical analysis of the essential oil by gas chromatography-mass spectrometry identified sesquiterpene hydrocarbons and oxygenated sesquiterpenes in bacupari leaves and fruits, and α-cedrene, α-chamigrene, α-trans-bergamotene, and β-curcumene as major compounds. Essential oil from leaves of G. gardneriana presented acaricidal activity on R. microplus (LC50 = 4.8 mg/mL; LC99 = 10.8 mg/mL) and larvicidal effect on A. aegypti (LC50 = 5.4 mg/mL; LC99 = 11.6 mg/mL), whereas essential oil from the fruits of G. gardneriana showed LC50 = 4.6 mg/mL and LC99 = 8.9 mg/mL against R. microplus and LC50 = 6.4 mg/mL and LC99 = 13.9 mg/mL against A. aegypti. These results thus demonstrate the potential acaricidal and larvicidal activity of essential oil of G. gardneriana, offering new perspectives for the realization of bioassays from this essential oil.