Drug resistance in Gram-negative bacteria is leading the public health towards a global crisis and is further compounded by the dearth in developing new antibiotics to treat these pathogens over the last five decades. Among the various proposed mechanisms of resistance development, exclusion of drug from bacterial cells by efflux pumps act as an efficient machinery expelling a broad range of antibiotics thereby lowering intracellular antibiotic concentration and rendering the available antibiotics obsolete. Among various classes of antibiotics, tetracyclines suffer resistance through efflux mechanism. Currently, resurrecting the obsolete antibiotic by using the non-antibiotic drugs/adjuvants has become one of the key successes to tackle the multi-drug resistant (MDR) Gram-negative bacterial infections. Herein, we report the sensitization of tetracycline with the help of a membrane active macromolecule, ACM-A_Hex against the resistant Gram-negative pathogens. ACM-A_Hex was found to exert its antibacterial action by depolarizing the membrane potential. Thereby increasing the accumulation of tetracycline in Gram-negative pathogens. The combination of the compound with tetracycline has demonstrated 4–128-fold potentiation in the antibacterial activity of the antibiotic. Overall, these in vitro studies have demonstrated immense potential of this combination to be further developed as therapeutic agents to treat Gram-negative bacterial infections.