AimThe objective of this study was to determine the efficacy and mechanisms of inactivation of two clinically relevant ESKAPE bacteria namely Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus by atmospheric pressure cold plasma. Methods and ResultsPlasma was generated between two brass grids by applying a radiofrequency electric field to a flow of helium. Intracellular generation of reactive species, alterations in cell membrane, and inactivation of bacteria in planktonic or biofilm growth were studied. Results were compared with commonly used antimicrobial drugs. Plasma exposure generated reactive oxygen and nitrogen species in bacteria, disrupted membrane integrity and reduced bacterial load. The efficacy in bacterial inactivation was comparable to antibiotics but exhibited a quicker killing rate. The antibacterial effect of plasma synergistically increased in association with antibiotics and did not diminish over repeated exposures, suggesting no development in bacterial resistance. ConclusionsThrough generation of reactive species, cold plasma altered cell membrane and effectively inactivated clinically important bacteria, both in suspension and in biofilms. Significance and Impact of the StudyAs cold plasma damages different targets in bacterial cells, it emerges as an effective strategy used alone or in combination with antimicrobial drugs to control microbial infections and prevent the selection of resistant bacterial strains.