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Nonthermal plasma generated by parallel-plate dielectric-barrier discharge with 60-kHz high-voltage power was used to sterilize the bacteria in atmospheric air. Two kinds of typical bacteria, gram-negative E. coli (ATCC8099) and grampositive S. aureus (ATCC6538), were used as test strains. Bacteria cells held by cover-glass were placed on the bottom electrode. By adjusting the applied voltage, gap spacing, and treatment time, the effects of plasma and electric field on bacteria inactivation were investigated. The transmission electron microscope was used to observe the damage of cells treated by plasma. The concentrations of K+, protein, and nucleic acid leaked from cells were measured for detecting the cytoplasm status after plasma treatment. Experimental results showed that almost 100% of S. aureus and E. coli strains were killed in less than 10- and 7-s plasma treatment, respectively. It is concluded that the reactive oxygen species (ROS) in plasma play a dominant role in the inactivation process but not the electric field. It is supposed that the ROS can oxidize the cell membrane and then damage the protein and nucleic acid inside the cells and, thus, kill the bacteria.