Aims The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated. Methods and Results The bacterial release of intracellular proteins, cytoplasmic beta-galactosidase and periplasmic beta-lactamase induced by T.maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T.maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium. Conclusions The effect of T.maroccanus EO on the outer membrane was comparable to that of PB, and both T.maroccanus EO and PB induce similar levels of beta-lactamase release. In addition, it also promoted the release of the cytoplasmic beta-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T.maroccanus EO. Significance and Impact of Study These results demonstrate that T.maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria.