Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant (MDR) opportunistic human pathogen, which produces an alternative penicillin-binding protein (PBP2a), rendering it resistant to β-lactam antibiotics. Therefore, the World Health Organisation has classified MRSA as a ‘high priority’ pathogen where novel antibacterial treatments are urgently required. The reversal of antimicrobial resistance by antibiotic adjuvants is a promising strategy to restore the activity of antibiotics against MDR bacteria. We have demonstrated that the zinc ionophore PBT2, alongside Zn (PBT2-Zn), can act as an antibiotic adjuvant, resensitising multiple MDR bacteria to killing by otherwise ineffective antibiotics. However, the molecular mechanisms underlying this resensitisation are currently unknown. To study this phenomenon, we investigated the ability of subtherapeutic PBT2-Zn to restore the bactericidal activity of the β-lactam oxacillin in vitro in the community-associated MRSA isolate USA300. We observed that PBT2-Zn combined with oxacillin dysregulated metal ion homeostasis in USA300 through the intracellular accumulation of zinc ions and reduced intracellular manganese ions. We also observed that manganese supplementation rescued USA300 from the bactericidal killing by PBT2-Zn-oxacillin treatment. The expression of several critical genes involved in β-lactam resistance were upregulated in response to PBT2-Zn-oxacillin, including mecA, which encodes PBP2a. In contrast, critical genes involved in virulence (e.g., saeS) and manganese transport (e.g., mntA) were downregulated. This study demonstrates that disruptions in bacterial metal ion homeostasis can trigger several genetic and biochemical changes in MRSA USA300, resulting in antibiotic sensitisation. Our investigations form the basis for further research to reveal the molecular and metabolic mechanisms that underpin antibiotic resistance reversal and how PBT2 can function as an adjuvant against diverse bacterial species. These data could enable us to design novel antibiotic adjuvants to sensitise a broad range of resistant bacteria to different antibiotic classes.