Background: Mushrooms with significant traditional medicine properties are rich sources of vitamins, proteins, fibres, minerals, and polyphenolic compounds. Objectives: Agaricus bisporus (edible white button mushroom) extracts and their purified polyphenolic content were examined in terms of cholesterol esterase (CEase) inhibitory activity. Methods: Organic solvent-based extraction methodologies were adopted to prepared the extracts. Two dimensional preparative thin layer chromatography (2D PTLC) technique and direct MS/ESI + method were also used to purify and elucidate the polyphenolic structures. Standard spectrophotometric analyses were employed to determine the flavonoid and phenolic acid contents as well as CEase inhibitory activity in both extract and purified polyphenols. Results:The findings supported the strong cholesterol esterase inhibitory activity of aqueous and methanolic extracts (separating funnel: 97.9 ± 1.1% and 97 ± 0.8%), and acetone extract (shake flask: 97.6 ± 0.2%). The investigation also documented the remarkable quantity of flavonoid (32.61 ± 4.1 mg/g tissue) and phenolic acid (12.38 ± 1.32 mg/g tissue). A fraction of strong blue colour phenolic acid was purified using 2D PTLC technique, and the direct MS/ESI + method adopted for the purified eluate also revealed the presence of p-hydroxy benzoic acid (m/z 136), ferulic acid (m/z 194), cinnamic acid (m/z 146), and protocatechuic acid (m/z 154). The purified phenolic acids represent a moderate cholesterol esterase inhibitory activity (24.7 ± 1.7%; IC50 = 2.61 µg). Conclusions:The study revealed the remarkable CEase inhibitory property, implying the cardioprotective attribute of A. bisporus.