Benzalkonium chloride (BC) is a commonly used disinfectant and preservative. This study describes changes in expression level at the transcriptomic and proteomic level for Escherichia coli K-12 gradually adapted to a tolerance level to BC of 7-8 times the initial MIC. Results from DNA arrays and two-dimensional gel electrophoresis for global gene and protein expression studies were confirmed by real-time quantitative PCR. Peptide mass fingerprinting by MALDI-TOF MS was used to identify differentially expressed proteins. Changes in expression level in adapted cells were shown for porins, drug transporters, glycolytic enzymes, ribosomal subunits and several genes and proteins involved in protection against oxidative stress and antibiotics. Adapted strains showed increased tolerance to several antibiotics. In conclusion, E. coli K-12 adapted to higher tolerance to BC acquired several general resistance mechanisms, including responses normally related to the multiple antibiotic resistance (Mar) regulon and protection against oxidative stress. The results revealed that BC treatment might result in superoxide stress in E. coli.