<p>Biofilm formation by methicillin-resistant <i>Staphylococcus aureus</i> is one of the most important issues causing persistent infections and resistance to complete eradication with antibiotic therapy. The challenges in managing biofilms have spurred the discovery of new anti-infective drugs derived from natural resources. In this study, we aimed to investigate the antibacterial and antibiofilm activities of the ethyl acetate fraction and the chalcone 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone isolated from <i>Cleistocalyx operculatus</i> (Roxb.) Merr. et Perry, Myrtaceae, against a clinical isolate of methicillin-resistant <i>Staphylococcus aureus</i>. Our results revealed that at the minimum inhibitory concentration value of 100&#xa0;µg/ml, the ethyl acetate fraction remarkably inhibited 90% of biofilm formation and disrupted nearly 50% of the mature biofilm of this pathogen. Additionally, 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone at subinhibitory concentrations of 100 and 50&#xa0;µg/ml reduced methicillin-resistant <i>Staphylococcus aureus</i> biofilm formation by 50.54 and 36.59%, respectively. Molecular docking and kinetic simulations revealed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone binds stably to the active site of sortase A, a key enzyme required for <i>Staphylococcus aureus</i> to form biofilm, via Pi-sulfur interactions with two conserved residues, Cys184 and Arg197. Spectral analysis further confirmed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone binding alters the structural conformation of sortase A. Taken together, our study provides novel evidence that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone isolated from the EtOAc fraction of <i>C. operculatus</i> could be a potential therapeutic candidate for combating methicillin-resistant <i>Staphylococcus aureus</i> infections.&#xa0;</p> Graphical Abstract <p></p>