To investigate the biological reactivity of plasma‐activated water (PAW), we prepared PAW using atmospheric air spark discharge over water at 4°C and 25°C, with Escherichia Coli (E. coli) as the microbial model. The biological reactivity of PAW at 25°C decreased rapidly by 3.32 Logs within 20 s of storage. At 4°C, PAW showed high reactivity within 20–30 s of storage but decreased by 3.50 Logs between 30 and 60 s. Our analysis revealed that PAW reactivity is dependent on short‐lived peroxynitrite (ONOOH), formed through H2O2 and HNO2 under acidic conditions. ONOOH actively reacts with amino acid functional groups, as measured by high‐resolution LC‐MS, likely contributing to the rapid inactivation of E. coli.