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In this study, we analyzed the essential oils (EOs) extracted by steam distillation from the leaves and flowers of Parthenium hysterophorus L., grown in three different locations in southern Ecuador: Espíndola (ESP), Loja (LOJ) and Quilanga (QUI). Approximately 97.45%, 98.27% and 95.99% of the oil constituents were identified using gas chromatography–mass spectrometry (GC-MS) and gas chromatography–flame ionization detection (GC-FID). Sesquiterpene hydrocarbons were predominant in the EOs. The most representative constituents of the sample from ESP were germacrene D (35.08%), myrcene (11.32%), (E)-β-ocimene (10.21%), (E)-caryophyllene (7.90%), germacra-4(15),5,10(14)-trien-1-α-ol (5.18%) and (E, E)-α-farnesene (4.99%), with an AChE IC₅₀ of 14.78 and with 16.38 and 93.16 µg/mL from ABTS and DPPH, respectively. The EOs from LOJ were characterized by the abundant presence of germacrene D (28.30%), myrcene (13.95%), (E)-β-ocimene (10.51%) and isolongifolan-7-α-ol (8.26%), with an AChE IC₅₀ of 16.65 and with 36.18 and 158.43 µg/mL from ABTS and DPPH, respectively. Finally, the EOs from QUI contained germacrene D (32.20%), myrcene (13.36%), (E)-β-ocimene (8.54%), (E, E)-α-farnesene (6.77%), germacra-4(15),5,10(14)-trien-1-α-ol (5.69%) and (E)-caryophyllene (5.37%), with an AChE IC₅₀ of 10.69 and with 14.50 and 92.98 µg/mL from ABTS and DPPH, respectively. This study identifies chemotypic variation in P. hysterophorus collected from different locations and, for the first time, the AChE inhibitor was evaluated. These findings provide a scientific basis for the clinical application of P. hysterophorus EOs in the future treatment of Alzheimer’s disease. Full article