The bactericidal mechanism of plasma activated water (PAW) is an important issue that needs to be addressed in plasma medicine. In this paper, the pH value and the concentrations of RONS (H2O2, [inline-formula], [inline-formula], OH·, ONOOH and 1O2) in PAW were measured by chemical and optical methods. The bacteria E. coli was used as a microbial model to study the bactericidal effects of PAW. It was found that the concentration of peroxynitrous acid (ONOOH) in PAW was too low to directly realize complete bactericidal effects of PAW. Actually, ONOOH further interacted with H2O2 to generate peroxynitric acid (O2NOOH), which eventually decomposed into superoxide anion radical ([inline-formula]·) and singlet oxygen (1O2); these two species enhanced the bactericidal effects of PAW substantially.