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Abstract: Enrichment of burdock leaf components and the inhibition effect of the obtained 7 burdock leaf fractions on formation of biofilm have been evaluated for the first time. Firstly, burdock leaf fractions were obtained though separation of burdock leaf extracts by macroporous resin column chromatography, and four fractions were obtained, namely, water elution fraction, 20%, 34% and 70% ethanol eluted fractions. It was found that the 20% and 34% ethanol fractions both significantly inhibited the formation of biofilm by Escherichia coli, with IC50 of 1.10 and 1.01 mg/ml, respectively. The 70% ethanol elution fraction exhibited the strongest inhibitory activity against biofilm formation with an IC50 of 0.017 mg/ml. In order to obtain more fractions with higher inhibitory activity, polyamide resin was used to further purify the 34% ethanol elution fraction. The second chromatography separation significant improved the activity of burdock leaf fractions and the IC50 of a new fraction was 0.03 mg/ml. The phenolic contents of the fractions ranged from 7.5 to 369.7 mg/g and there was a high correlation between the anti-biofilm capacity and phenolic contents, except for 70% ethanol fraction. Scanning electron microscopy (SEM) observation showed that there was no biofilm formation for cultures treated with burdock leaf fraction, conforming the significant inhibition activity of burdock leaf fraction against the formation of biofilm. The results suggest that separation and enrichment of the active ingredients could significantly improve the activities of fractions and the obtained burdock leaf fractions provide new alternatives for control of biofilms. [Copyright &y& Elsevier]