The rapid proliferation of multidrug-resistant (MDR) bacterial pathogens poses a serious threat to healthcare worldwide. Carbapenem-resistant (CR) Enterobacteriaceae, which have near-universal resistance to available antimicrobials, represent a particularly concerning issue. Herein, we report the identification of AMXT-1501, a polyamine transport system inhibitor with antibacterial activity against Gram-positive and -negative MDR bacteria. We observed minimum inhibitory concentration (MIC)/MIC values for AMXT-1501 in the range of 3.13-12.5 μM (2.24-8.93 μg /mL), including for methicillin-resistant (MRSA), CR , , and . AMXT-1501 was more effective against MRSA and CR than vancomycin and tigecycline, respectively. Subinhibitory concentrations of AMXT-1501 reduced the biofilm formation of and . Mechanistically, AMXT-1501 exposure damaged microbial membranes and increased membrane permeability and membrane potential by binding to cardiolipin (CL) and phosphatidylglycerol (PG). Importantly, AMXT-1501 pressure did not induce resistance readily in the tested pathogens.