The significant decline in novel antibiotic discovery and commercialisation has paralleled an escalation in antibiotic resistance, highlighting the urgent need for new antibiotic development and complementary therapy. Today, several strategies are being investigated to combat bacterial resistance to existing antibiotics, including the repurposing of existing drugs, originally developed as therapeutics for non-infectious disease. Here we report the synergy between ionophore-induced physiological stress in Gram-positive and Gram-negative bacteria and antibiotic treatment. PBT2 is a safe-for-human-use transition-metal ionophore that has progressed to Phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. Our data shows that PBT2 disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and pan-drug resistant Klebsiella pneumoniae. While ineffective alone against resistant bacteria, several clinically-relevant antibiotics act synergistically with PBT2 to enhance the killing of these resistant pathogens. We were unable to select for any mutants resistant to PBT2. Furthermore, we have generated proof-of-principle data demonstrating that PBT2 restores antibiotic sensitivity in multi-drug resistant Gram-positive and Gram-negative pathogens in vivo during wound and systemic infection respectively. GAS, MRSA, VRE and K. pneumoniae are common causative agents of hospital- and community-acquired infection, and are major contributors to global human mortality and morbidity. This novel therapeutic strategy represents a new paradigm in the fight against antibiotic resistant pathogens, whereby disruption of bacterial cellular homeostasis reverses antibiotic resistant phenotypes in a number of priority human bacterial pathogens.