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To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti-biofilm mechanisms.Anti-biofilm activity of the plant materials on clinical isolated of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staph. aureus was employed using a crystal violet-stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml−1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti-biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra-MIC, MIC and sub-MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall.The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti-biofilm activity.This evidence highlighted the anti-biofilm potency of the natural products and clarified their anti-biofilm mechanisms.