Quaternary ammonium compounds (QACs) benzalkonium (BZK) and cetrimide (CET) are common disinfectants used to inhibit or eradicate Gram-negative bacteria in clinical and agricultural products. QAC tolerance in Escherichia coli and other Enterobacterales species can confer cross-resistance to various clinically used antibiotics, making it important to understand mechanisms of QAC tolerance in greater depth. QAC adaptation by E. coli is hypothesized to alter MarRAB regulated genes that converge on the outer membrane, specifically, lipid A biosynthesis and transport genes, porins, and efflux pump systems. To test this, we performed a 'multi'-omics and phenotypic characterization of E. coli K-12 adapted to BZK and CET, to assess how QACs alter cell growth, genomics, and proteomics. E. coli adapted to either BZK and CET resulted in strains with stable QAC tolerance when either drug was omitted, elongated and narrower cell morphologies by scanning electron microscopy, and reduced growth fitness when compared to un-adapted E. coli. Antimicrobial susceptibility testing revealed that QAC adaptation increased E. coli tolerance by >4-fold to BZK, CET, and other QACs but no antibiotic cross-resistance was observed. Single nucleotide variants identified by whole genome sequencing and differentially accumulated proteins by liquid chromatography-mass spectrometry identified alterations to various QAC-adapted E. coli genes and proteins belonging to: lipid A biosynthesis and transport (lpxLM, msbA, mlaACF), the mar-sox-rob regulatory pathway (marR, rob), DNA/protein translation (gyrA, rpsA, rpoB, rapA). These alterations validate the hypothesis that mar-sox-rob network plays a role in QAC tolerance and identifies additional stress inducible genetic and protein QAC tolerant biomarkers.