Aim Analysing the effect of step-wise exposure of Escherichia coli isolates to sublethal concentrations of triclosan (TCS) that is widely used as an antiseptic, preservative and disinfectant. Methods and Results Changes in the tolerance to the biocide itself and the cross-resistance to clinically important antibiotics were analysed. The involvement of efflux mechanism was studied as well as the possible implication of modifications in cytoplasmic membrane properties including integrity, permeability, potential and depolarization in the resistance mechanisms. Most of E. coli isolates that were adapted to TCS showed increased antimicrobial resistance, lower outer and inner membrane permeability, higher membrane depolarization, more negative membrane potential and enhanced efflux activity using qRT-PCR. Nonsignificant change in membrane integrity was found in the adapted cells. Conclusion This study suggests that the extensive use of TCS at sublethal concentrations contributed to the emergence of antibiotic resistance in E. coli clinical isolates, by inducing changes in bacterial membrane properties and enhancing the efflux system. Significance and Impact of the Study: The extensive usage of TCS has a deleterious effect on the spread of antibiotic resistance, and more studies are needed to investigate the molecular mechanisms underlying the effects of TCS.