Disinfection of food contact surfaces is a challenging task, aggravated by bacteria's capacity to survive and/or resist antimicrobials by means of mechanisms not yet completely understood. This work evaluated the susceptibility of Listeria monocytogenes and Salmonella enterica biofilms to four disinfectants, and analyzed how those chemical agents influenced stress-response and virulence genes expression by surviving cells. Three strains of each bacterial species mentioned were used, and their biofilms were treated with sodium hypochlorite, benzalkonium chloride, hydrogen peroxide, and triclosan using the Calgary Biofilm Device. Expression of L. monocytogenes and S. enterica stress-response genes cplC and ropS, and virulence genes prfA and avrA, respectively, was analyzed through quantitative real-time polymerase chain reaction. Results showed sodium hypochlorite to have the lowest minimum biofilm eradication concentration values (3.125 μg/ml), whereas triclosan had the worst performance since no S. enterica biofilm eradication was achieved even at the maximum concentration used (4,000 μg/ml). L. monocytogenes stress-response gene and S. enterica virulence gene were significantly upregulated in surviving cells compared with controls. In general, this work points out sodium hypochlorite as the most effective disinfectant against biofilms of both species used, and L. monocytogenes biofilms to be more susceptible to disinfection than S. enterica biofilms. Moreover, it was found that disinfection surviving biofilm cells seem to develop a stress response and/or become more virulent, which may compromise food safety and potentiate public health risk.