Sanitizing effect of ethanol on a Yersinia enterocolitica biofilm was evaluated in terms of biomass removal and bactericidal activity. We found that 40 % ethanol was most effective for biofilm biomass removal; however, no significant difference was observed in bactericidal activity between treatment with 40 and 70 % ethanol. This unexpected low ethanol concentration requirement for biomass removal was confirmed using biofilms of two additional pathogenic bacteria, Aeromonas hydrophila and Xanthomonas oryzae. Although only three pathogenic Gram-negative bacteria were tested and the biofilm in nature was different from the biofilm in this study, the results in this study suggested the possible re-evaluation of the effective sanitizing ethanol concentration 70 %, which is the concentration commonly employed for sanitization, on bacteria in a biofilm.