Aims This study aimed to evaluate the effect of a fraction of burdock (Arctium lappa L.) leaf on the initial adhesion, biofilm formation, quorum sensing and virulence factors of Pseudomonas aeruginosa. Methods and Results Antibiofilm activity of the burdock leaf fraction was studied by the method of crystal violet staining. When the concentration of the burdock leaf fraction was 20mgml(-1), the inhibition rates on biofilm formation of P. aeruginosa were 100%. The burdock leaf fraction was found to inhibit the formation of biofilm by reducing bacterial surface hydrophobicity, decreasing bacterial aggregation ability and inhibiting swarming motility. Interestingly, the burdock leaf fraction inhibited the secretion of quorum-sensing (QS) signalling molecule 3-oxo-C12-HSL and interfered quorum sensing. Moreover, the QS-regulated pyocyanin and elastase were also inhibited. Chemical composition analysis by UPLC-MS showed 11 active compounds in the burdock leaf fraction. Conclusions The burdock leaf fraction significantly inhibited the formation of biofilm and quorum sensing, as well as significantly decreased the content of virulence factors. Significance and Impact of the Study This study introduces a natural and effective bacterial biofilm inhibitor, which could also significantly decrease the content of virulence factors and the drug resistance of P. aeruginosa.