Prunus persica fruit is a source of anti-cholinesterase agents and led to an increase of acetylcholine levels in brain tissue as a usefull tool in the Alzheimer’s disease therapy. This study aimed to propose a convenient method for the purification of cholinesterase inhibitors from P. persica water extract. Water extract from the fruit was ultrafiltered (0.2 μm→5 kDa→3 kDa) followed by preparative chromatography (Sephadex LH-20, high pressure C18) and high pressure analytical C18 chromatography. The chemical structures of inhibitors were confirmed using infrared and 1H-NMR spectroscopies. The anti-acetylcholinesterase activity was measured using the colorimetric method in fractions obtained after each stage of purification. Polyphenolic cholinesterase inhibitors identified in peach fruit were kaempferol, quercetin and quercetin-3-O-rhamnoglucoside (rutin). The relatively fast purification procedure elaborated in this work can be adopted for the isolation of phenolic cholinesterase inhibitors from fruit extracts related to P. persica fruit.