Aims: Nonthermal plasma agents (reactive species and charged particles) are generally generated together. Previous studies of nonthermal plasma agents did not investigate the role of a microbial inactivation agent without interference from other agents. Consequently, the exact mechanism underlying their activity remains unclear. The present experiment was conducted to study the mechanism underlying Escherichia coli inactivation by nonthermal plasma. Methods and Results: The mechanism underlying E. coli inactivation by charged particles was studied using pure argon plasma. Results showed that cell wall damage owing to strong electrostatic forces did not occur during direct current (DC) plasma treatment with Ar or N-2. Next, the inactivation effects of excited N-2(*), N-2(+), ozone, OH radicals, and nitric oxide were investigated using pure nitrogen plasma and air plasma. Morphological changes and cell rupture of E. coli were observed after 5 min of treatment with nonthermal plasma in air, but not with argon and nitrogen plasma treatments. Conclusions: Our data demonstrate that reactive oxygen species play an essential role in the inactivation of E. coli. Significance and Impact of the Study: A clear understanding of the mechanisms underlying nonthermal plasma's inactivation of micro-organism is essential for the practical applications of nonthermal plasma techniques.