The formation of biofilms play crucial role in development of multiple drug resistance (MDR) in microorganisms and makes treatment more complex. Therefore, it is necessary to employ a strategy that effectively inhibits the formation of biofilm to treat MDR microorganisms. In the present studies, anti-biofilm activity of the methanolic extract of Terminalia arjuna stem bark (MeOH-TASB) and its purified flavonoids was evaluated against human pathogenic bacteria Staphylococcus aureus and Escherichia coli. The MeOH-TASB and its constituent flavonoids (baicalein and quercetin) showed potent antibacterial activity at relatively higher concentrations and inhibited biofilm formation at lower concentrations in both S. aureus and E. coli. Apart from inhibiting the formation of biofilm, MeOH-TASB, baicalein and quercetin disrupted pre-formed biofilms. These results merit further investigation of the potential of MeOH-TASB, baicalein and quercetin for the treatment of bacterial infections.