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Abstract Microbial biofilms are of critical concern because of their recalcitrance to antimicrobials. Cold atmospheric plasmas (CAP) represent a promising biofilm remediation strategy as they generate reactive oxygen and nitrogen species (RONS), but mechanisms underpinning CAP‐biofilm interactions remain unknown. We assess the impact of treatment modality on biofilm inactivation and show that CAP killing ofStaphylococcus aureusbiofilms is dependent on treatment conditions, including solution chemistry. In dry treatments, biofilms are locally ablated due to plasma‐produced O flux. For saline‐submerged biofilms, while we show that ClO−is generated at high concentrations in larger treatment volumes, CAP inactivation at low ClO−concentrations implicates other reaction pathways. Finally, we demonstrate CAP efficacy over conventional antimicrobials, underscoring its promise as a biofilm treatment approach.more » « less
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