Deadly diseases caused by pathogenic bacteria and viruses have increasingly victimized humans; thus, the importance of disinfection has increased in medical settings as well as in food and agricultural industries. Plasma contains multiple bactericidal agents, including reactive species, charged particles, and photons, which can have synergistic effects. In particular, the chemicals formed in aqueous solution during plasma exposure have the potential for high antibacterial activity against various bacterial infections. Here, we report the antibiotic potency of plasma-treated water (PTW). To illustrate the applicability of PTW for disinfecting biological substances, an Escherichia coli biofilm was used. We sought to identify the chemical species in PTW and investigate their separate effects on biofilm removal. Dielectric barrier discharge in ambient air was used to prepare the PTW and treat the biofilm directly. Hydrogen peroxide, ozone, and nitrites were identified as the long-lived reactive species in the PTW, whereas hydroxyl radicals and superoxide anions were identified as the short-lived reactive species in the PTW; all these species showed an ability to disinfect in biofilm removal.