We genetically analyzed the roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12 BW25113. We used 22 mutants missing various genes related to the multidrug efflux pumps and found that the biofilm formation of emrD, emrE, emrK, acrD, acre and mdtE-deleted mutants was extremely decreased. These results indicate that some multidrug efflux pumps significantly contribute to the biofilm formation of E. coli.