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Siparuna muricata is an aromatic native shrub or tree from Ecuador known as “limoncillo” or “limón de la sierra” due to its citrus odor. In this study, the chemical composition and biological activity of essential oil from the leaves and fruits of this species was determined. The essential oil was isolated by subjected to hydrodistillation. The chemical composition was determined by gas chromatography equipped with a flame ionization detector and gas chromatography coupled to mass spectrometry. The enantiomeric distribution was determined by gas chromatography using an enantioselective column. The antimicrobial activity against three Gram-positive bacteria, two Gram-negative bacteria and two fungi was determined by the broth microdilution method. The antioxidant activity was analyzed using the 2,2-diphenyl-1-picrylhydryl free radical and 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation scavenging activity of essential oil. The acetylcholinesterase inhibitory effect of the essential oil was measured using a spectrophotometric method. The yield was 1.2 ± 0.1 mL/kg for leaves and 1.8 ± 0.2 mL/kg for fruits. A total of 51 compounds were identified in the leaves of the essential oil and 41 in the fruits. In both cases, the chemical compositions were dominated by the group of monoterpene hydrocarbons compounds. The main compound was α-pinene with 23.22 ± 1.03% in essential oil of the leaves and limonene with 24.92 ± 1.20% in the fruits. In both essential oils, five pairs of enantiomers with different enantiomeric excesses were identified. The essential oil of limoncillo leaves presented a strong activity against the fungus Aspergillus niger (ATTC 10231) and Gram-positive bacterium Enterococcus faecium (ATCC 27270) with a MIC of 250 μg/mL and 500 μg/mL, respectively. The essential oil from fruits and leaves of Siparuna muricata presented a moderate antioxidant activity with the ABTS method with a SC50 of 775.3 ± 1.3 µg/mL and 963.3 ± 1.6 µg/mL, respectively. Additionally, the leaves essential oil reported an IC50 value of 52.98 ± 1.04 µg/mL and the fruits essential oil an IC50 value of 98.84 ± 1.04 µg/mL, which can be considered a very strong anticholinesterase activity.