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Background: The role of hospital disinfectants in infection prevention is paramount; however, some studies indicate that sublethalexposure may enhance resistance to lethal concentrations of an antibiotic through cross-resistance mechanisms. The goal of this research is to determine the molecular mechanisms involved in the antibiotic resistance of biofilm-formingEscherichia coli caused by disinfectants, which were collected in neonatal care units. Methods: We studied35 strainsfrom nurserybabiesthrough specific molecular, biochemical, and phenotypic techniques. The strains underwent exposure to gradient concentrations of quaternary ammonium compounds (QACs), chlorhexidine, ethanol, and sodium hypochlorite. The overrepresented patterns of cross-resistance,biofilm formingability, as well as molecular and genetic mechanisms, were assessed through QPCR, biofilm assays, and antibiotic susceptibility testing. Results: Treatment of QACs resulted in an increase in biofilm formation by 20.0% (p<0.001) and an increase in the prevalence of multidrug resistance (MDR) from 52.9% to 64.7%. Exposure to chlorhexidine resulted in 8.7% increased biofilm formation as well as a 5.9% increase in β-lactam resistance. On the contrary, ethanol and sodium hypochlorite not only reduced biofilm formation by 33.3% and 25.6% respectively, but also reduced the prevalence of MDR to 47.1%. Molecular studies confirmedhypothesisof overexpression of acrAB-tolC efflux pump biofilm related to quorum sensing (csgA, bssR) genes under the influence of QACs. There is also a strong correlation between biofilm density (OD₅₇₀ ≥1.25) and resistance to carbapenem (r=0.78, p<0.001). Conclusions: It has been established through this study that both QACs and chlorhexidine are able to trigger cross-resistance through mechanisms that are biofilm dependent. In contrast, oxidative disinfectants are able to eliminate and control bioburden without the potential of encouraging resistance.Such findings directly inform policies regarding hospital sanitization procedures and programs aimed at the minimization of antimicrobialagents’usage.