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While cold atmospheric plasma (CAP) is known for its bactericidal effects, its impact on biofilm architecture and physiology, and subsequent antimicrobial susceptibility remains poorly understood. This study investigates the effects of CAP treatment on S. aureus biofilms. We demonstrate that CAP not only reduces biofilm but also disrupts biofilm architecture and alters bacterial cell morphology. Transcriptomic analysis reveals distinct gene expression profiles following treatments with CAP and hydrogen peroxide, one of its active components, highlighting unique stress responses. We further show that CAP treatment of S. aureus grown on agar plates sensitizes bacteria to additional CAP exposure and induces genomic alterations involved in cell envelope integrity, defence mechanisms and DNA replication, recombination, and repair. Importantly, CAP treatment enhances bacterial susceptibility to the topical antibiotic for wound care mupirocin. 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