Aim A lectin Concanavalin A (ConA) derived from Canavalia ensiformis (jack bean) exhibits high-binding affinity to carbohydrates on bacterial cell surfaces. The objective of this study was to inhibit the biofilm formation of the foodborne pathogens enterohemorrhagic Escherichia coli and Listeria monocytogenes using ConA prepared by a membrane-based extraction method. Methods and Results ConA was extracted using a simple and inexpensive membrane method instead of a chromatography approach. The extracted ConA was effective in inhibiting biofilms of E. coli by 30-fold and L. monocytogenes by 140-fold. In addition, ConA decreased the swimming motility of enterohemorrhagic E. coli EDL933 (EHEC) by 37%, resulting in low biofilm formation, as ConA binding to the bacterial cell surfaces might cause a reduced capability to adhere due to low cellular motility. We confirmed that the extracted ConA contains active components at less than 10 kDa as well as ConA multimers (>30 kDa) that repress EHEC biofilms. Additionally, noncell-based mannose reduced the activity of ConA in inhibiting biofilms. Conclusions ConA extracted using the membrane-based method is active in inhibiting the biofilm formation by E. coli and L. monocytogenes via the mannose-binding affinity of ConA. Significance and Impact of the Study ConA can be used as a promising anti-adherent and antibiofilm agent in inhibiting biofilm formation by enterohemorrhagic E. coli and L. monocytogenes. The membrane-based extraction approach may be applied for the economic production of biologically active lectins.