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Aims Our aim was to assess the effects of step-wise exposure to didecyl dimethyl ammonium chloride (DDAC) on the antimicrobial (antibiotics and biocides) susceptibilities of food-associated bacterial strains. Methods and Results Adaptive responses of bacterial strains were investigated by exposing the strains daily to increasing sub-inhibitory concentrations of DDAC for 7 days. Following adaptation to DDAC, a 3-fold increase in the MIC values for this biocide was observed in 48% of the Escherichia coli and Listeria monocytogenes strains, and 3% of the Salmonella strains. Reduced susceptibility to other biocides was found with the most important increase in MIC for benzalkonium chloride (BC) and a commercial biocide formulation (Galox Horizon) containing DDAC and glutaraldehyde, for all species except Salmonella. Increase in antibiotic MIC values was more pronounced in Escherichia coli in terms of antibiotic numbers and of magnitude (from 4- to 32-fold increase) and, to a lesser extent, in Salmonella strains. Most of these strains had acquired resistance to ampicillin, cefotaxime, ceftazidime, chloramphenicol and ciprofloxacin. Conclusions The effects of exposure to DDAC on biocides and antibiotics susceptibilities depend upon the bacteria species. Significance and Impact of the Study Extensive use of DDAC at sub-inhibitory concentrations may lead to the development of antibiotic-resistant bacteria and may represent a public health issue This article is protected by copyright. All rights reserved.