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Flavonoids are secondary plant metabolites that have long been included in the human diet. These natural products are well known for their beneficial effects on health. Thus, in this study, the in vitro inhibitory activity of 28 different flavonoids (1-28) against acetylcholinesterase was evaluated, and the type of inhibition for the most active compound (12) was studied. Acetylcholinesterase (AChE) inhibitors are considered the main class of medication used for the treatment of the primary dementia phase in Alzheimer’s disease (AD). Our study used galantamine as a positive control and showed that 7, 8-dihydroxyflavone (12) has the highest anti-acetylcholinesterase activity. The inhibitory type of 7, 8-dihydroxyflavone (12), which is non-competitive inhibition, was determined for the first time. Furthermore, the obtained data allowed us to identify the characteristic flavonoid structure that facilitates AChE inhibition: the presence of a hydroxyl group at C7 in the benzo-γ-pyrane ring of the molecule.