Aloysia polystachya is used as a sedative and antidepressant by the indigenous populations of Argentina and Paraguay, but the compounds associated with these activities have not been determined. We have separated and identified the main constituents of the hydroethanolic extract of A. polystachya by ultra-performance liquid chromatography-mass spectrometry and confirmed the presence of acteoside, isoacteoside, 6'-acetylacteoside, and 4’,4’’’,5,5’’-tetrahydroxy-6,6’’,3’’’-trimethoxy-[C7–O–C7’’]-biflavone by NMR spectroscopy. Inhibitory activities of the hydroethanolic extract and purified phenylethanoid glycosides against monoamine oxidase-A were assessed using a standard fluorometric assay. The hydroethanolic extract inhibited monoamine oxidase-A activity in a dose-dependent manner with an IC50 of 9.2 µg/mL, while the selective monoamine oxidase inhibitor clorgyline exhibited an IC50 of 0.06 µg/mL (0.22 µM). Acteoside was the strongest inhibitor of monoamine oxidase-A (IC50 value of 5 µM), whereas isoacteoside and 6'-acetylacteoside showed IC50 values of around 10 µM. The results showed that phenylethanoids from a hydroethanolic extract of A. polystachya have been found to have inhibitory activity against monoamine oxidase-A. It is likely that the mode of action of the acteosides is multi-targeted, involving the downregulation of inflammatory molecules and neutralization of oxidation reactions as well as inhibition of monoamine oxidase-A.