Antimicrobial resistance (AMR) is a critical threat to human health. In recent decades, the Gram-positive bacteria Enterococcus faecium and Staphylococcus aureus have acquired broad-spectrum resistance to several major classes of antibiotics. With the rise of AMR concurrent with a reduction in novel drug discovery, new treatments are urgently needed. One promising source of new antimicrobials are the ion transporter compounds, termed ionophores, which have demonstrated broad-spectrum antimicrobial activity. Here, we sought to identify novel ionophores and characterise their antimicrobial properties. One compound, designated “704,” exhibited antimicrobial activity across a panel of multidrug-resistant (MDR) strains of E. faecium and S. aureus with activity in the low (1 – 4) µg/ml range with dose-dependent bactericidal activity against selected strains. In addition, 704 was found to be non-toxic to mammalian cells in vitro. Bacterial resistance was not detected following 30 consecutive days of serial passage of bacterial growth. Finally, 704 demonstrated efficacy in reducing the burden of MDR S. aureus in a murine wound infection model and effectively perturbed metal ion homeostasis in treated bacteria. Taken together, 704 is a promising candidate for further investigation to treat Gram-positive MDR pathogens.