In 2018, antibiotic resistant bacterial infections were the source of $2 billion (U.S.) in medical bills in the United States alone. With rising antibiotic resistance, prevention of infection by chemical disinfection prior to the development of an infection is preferred over treating an infection. Production of typical chemical disinfectants, however, carries its own risks, including environmental damage; thus, endogenous cellular oxidants have become an attractive choice for disinfection. For example, peroxynitrite (ONOOH), an endogenous cellular oxidant, is produced in cells during apoptosis and causes oxidative damage to cell membranes, proteins, and DNA. If ONOOH could be produced in a reproducible and environmentally friendly manner, it could be a good candidate for an effective disinfection agent. Plasma-activated water (PAW), produced by exposing water to cold atmospheric plasma, has been shown to have antibacterial properties that are believed to rely on reactive oxygen and nitrogen species (RONS) in solution (e.g. nitrate, nitrite, and hydrogen peroxide) which may ultimately form peroxynitrite. The present study determines bactericidal effects of PAW-produced RONS in isolation and in concert in order to understand the impact of peroxynitrite in PAW’s antibacterial properties. A fluorescent probe for peroxynitrite using folic acid selectively quantified peroxynitrite in mimicked PAW solutions and in PAW. Mimicked PAW solutions that form peroxynitrite prove to be almost as effective as PAW at killing Escherichia coli bacteria (E. coli). Another probe using salicylic acid quantified the hydroxyl radicals in solution. As OH is a degradant of peroxynitrite, these experiments confirm the presence of the bactericidal species ONOOH. Studies of aged PAW demonstrate that the peroxynitrite concentration, and concomitantly the antibacterial effect of PAW, decreases over a short amount of time. Overall, these results clearly indicate the possibility of PAW being an environmentally friendly disinfectant that can be produced at the point of use.