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Ageratina dendroides is an aromatic species native to Ecuador. In this study, the chemical composition, enantiomeric distribution, and antifungal, antibacterial, antioxidant, and anticholinesterase activities of the essential oil isolated from aerial parts of Ageratina dendroides were determined. The quantitative chemical composition was determined using gas chromatography equipped with a flame ionization detector. The qualitative chemical composition was determined using gas chromatography coupled with mass spectrometry. The enantiomeric distribution was determined using an enantioselective chromatographic column. A broth microdilution method was used to determine the antibacterial activity. This antibacterial activity was tested against three Gram-negative bacilli bacteria and three Gram-positive cocci bacteria. The antifungal activity was tested against two fungi, a fungus, and a yeast. The antioxidant activity was determined using the ABTS (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) method and DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The anticholinesterase activity was analyzed using the spectrophotometric method. Sixty-eight volatile compounds were identified in the essential oil. These compounds represent 99.49% of the total composition. In terms of the number of compounds and their relative abundance, the most representative group was sesquiterpene hydrocarbons, with thirty-four compounds and an abundance of 49.22%. The main constituents were found to be andro encecalinol (14.41 ± 1.26%), germacrene D (8.86 ± 1.06%), amorpha-4,9-dien-14-al (7.68 ± 0.70%), β-sesquiphellandrene (7.01 ± 1.49%), α-muurolol (5.89 ± 0.93%), and 7-epi-α-selinene (5.68 ± 0.53%). Five pairs of enantiomers were identified in the essential oil of Ageratina dendroides. The essential oil did not report antimicrobial activity at the maximum concentration tested (MIC > 4000 µg/mL) against any of the microorganisms tested. The antioxidant activity of the essential oil was strong in the ABTS method, with an SC50 of 33.2 ± 1.4 µg/mL. Additionally, the Ageratina dendroides essential oil reported moderate anticholinesterase activity, with an IC50 of 297.8 ± 1.03 µg/mL.