Whey is a major by-product of cheese manufacture and contains many valuable constituents, such as β-lactoglobulin, lactoferrin and immunoglobulin. The current study determined the anti-biofilm activity of bioconversion of whey by Lactobacillus plantarum (LP-W), L. rhamnosus GG (LR-W), L. brevis (LB-W) and Enterococcus faecium (EF-W) against foodborne pathogenic bacteria, Escherichia coli O157:H7 and Listeria monocytogenes. When the foodborne pathogenic bacteria were co-incubated with LP-W, LR-W, LB-W or EF-W, biofilms by E. coli O157:H7 and L. monocytogenes were significantly reduced by all bioconversion of whey. Moreover, LP-W, LR-W, LB-W and EF-W also dramatically reduced pre-formed biofilm by E. coli O157:H7 and L. monocytogenes, suggesting that the bioconversion of whey effectively suppress the development and disruption of biofilm by foodborne pathogenic bacteria. Furthermore, in order to determine the growth kinetics of E. coli O157 and L. monocytogenes planktonic cells in the presence the bioconversion of whey, LP-W, LR-W, LB-W and EF-W did not significantly inhibited the growth of foodborne pathogenic bacteria, implying that the bioconversion of whey reduces the biofilm without the decrease of bacterial growth. Taken together, these results suggest that bioconversion of whey by lactic acid bacteria could be a promising agent for the reduction of microbial biofilm.