Acetoacetate (AAA) was identified as a biofilm inhibitor in a previous study, where the effect of 190 carbon and nitrogen sources on biofilm amounts by Escherichia coli O157:H7 was determined. With this study, we tested the effect of AAA on growth and biofilm amounts of Cronobacter sakazakii, Serratia marcescens and Yersinia enterocolitica. AAA reduced growth and biofilm amounts of the three pathogens, albeit at rather high concentrations of 10 to 35 mg ml-1 . Acetoacetate at a concentration of 5 mg ml-1 reduced Y. enterocolitica mRNA transcripts of the flagellar master regulator operon flhD, the invasion gene inv, and the adhesion gene yadA. Transcription of the regulator of plasmid-encoded virulence genes virF, the plasmid-encoded virulence gene yopQ, and ymoA were largely unaffected by AAA. Importantly, AAA did not cause an increase in transcription of any of the tested virulence genes. As a more cost efficient homologue of AAA, the effect of ethyl acetoacetate (EAA) was tested. EAA reduced growth, biofilm amounts and live bacterial cell counts up to 3 logs. IC50 values ranged from 0·31 mg ml-1 to 5·6 mg ml-1 . In summary, both AAA and EAA inhibit biofilm, but EAA appears to be more effective.Bacterial biofilms are communities of bacteria that form on surfaces and are extremely difficult to remove by conventional physical or chemical techniques, antibiotics or the human immune system. Despite advanced technologies, biofilm still contributes to 60 to 80% of human bacterial infections (NIH and CDC) and cause problems in many natural, environmental, bioindustrial or food processing settings. The discovery of novel substances that inhibit biofilm without increasing the virulence of the bacteria opens doors for countless applications where a reduction of biofilm is desired.