The direct inactivation effects of an atmospheric pressure direct current (DC) air plasma against planktonic Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in aqueous solution are investigated in vitro. Upon plasma treatment, extensively analyses on cell culturability, metabolic capacity, membrane integrity, surface morphology, cellular proteins, nucleic adds and intracellular reactive oxygen species (ROS) for both bacterial species were carried out and significant antimicrobial effects observed. Compared with the cellular culturability, a sub-lethal viable but non-culturable (VBNC) state was induced while more S. aureus entered this state than E. coli. Damaged bacterial outer structures were observed and the total concentrations of cellular protein and nucleic acid decreased for both bacteria after plasma treatment. The plasma-induced aqueous reactive species (RS) and intracellular ROS might produce detrimental effects to the bacteria, while S. aureus was less susceptible to the discharge after a 20-min exposure compared to E. coll. (C) 2018 Elsevier B.V. All rights reserved.