This study investigated the effect and possible mechanism of berberine on biofilm formation by methicillin-resistant Staphylococcus aureus. Compared with chloramphenicol, berberine showed a significantly stronger inhibitory effect on the growth of methicillin-resistant Staphylococcus aureus, and the minimum inhibition concentration and minimum bactericidal concentration for berberine were 0.05 mg/ml and 0.1 mg/ml, respectively (p< 0.05). In addition, berberine significantly impaired biofilm formation and reduced biofilm thickness in a dose-dependent manner (p< 0.05). Furthermore, the results of quantitative real-time polymerase chain reaction revealed that, at concentrations ranging from 20 to 120 μg/ml, berberine inhibited the expression of agrA, agrD, agrB, and agrC(p< 0.05). The molecular regulatory mechanism of berberine inhibition may be obtained by its downregulation of the expression of agrA, agrD, agrB, and agrC, thereby inhibiting biofilm formation by methicillin-resistant Staphylococcus aureus. These findings demonstrate that berberine has a great potential as a quorum sensing inhibitor against biofilm-associated infections.