Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)-<i>cis</i>-<b>1</b>, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of <b>1</b> by HPLC and (ii) preparing two close achiral analogues of <b>1</b>, i.e., compounds <b>4</b> and <b>6</b>. An eudismic ratio as high as 20 was observed for the (-) enantiomer of <i>cis</i>-<b>1</b>. The X-ray crystal structure of the complex between the (-)-<i>cis</i>-<b>1</b> eutomer (coded as <b>MC1420</b>) and <i>T. californica</i> AChE was determined at 2.8 Å, and docking calculation results suggested that the eutomer in (1<i>R</i>,3<i>S</i>) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1<i>S</i>,3<i>R</i>) configuration. The achiral analogues <b>4</b> and <b>6</b> were less effective in inhibiting AChE compared to (±)-<i>cis</i>-<b>1</b>, but interestingly butylamide <b>4</b> emerged as a potent inhibitor of butyrylcholinesterase (BChE).