"Cold atmospheric plasma (CAP), a non-thermal ionised gas producing reactive oxygen and nitrogen species (RONS), has emerged as a promising adjuvant to antibiotics for disrupting biofilms and modulating tissue responses. In a series of transnational studies across Ireland, the UK, and the US, we investigated CAP’s role in reprogramming infection microenvironments. Short exposures (90–120 s) to CAP significantly enhanced the susceptibility of Staphylococcus aureus and Pseudomonas aeruginosa biofilms to antibiotics, achieving up to 512-fold reductions in MBEC. These synergistic effects were not attributable to direct killing alone. Real-time structural and lipidomic profiling (REIMS, fluorescence imaging) confirmed biofilm matrix collapse and phospholipid depletion. Transcriptomic analyses revealed oxidative stress responses and membrane-associated gene upregulation, consistent with transient outer membrane disruption and increased antimicrobial penetration. Building on these in vitro findings, we assessed CAP’s effects in vivo. In a sterile muscle injury model, CAP induced rapid neutrophil and mast cell recruitment, antioxidant gene expression, and sustained activation of pro-regenerative programs, promoting myogenesis over adipogenesis. In contrast, surgical revision of S. aureus implant infections alone exacerbated virulence factor expression and triggered a multi-tissue inflammatory response without reducing bacterial burden. These findings demonstrate that CAP not only sensitises biofilms to antimicrobials but also modulates host inflammatory and repair pathways. This dual antimicrobial–immunomodulatory activity supports its development as a non-antibiotic, stewardship-aligned intervention for recalcitrant infections."