Pseudomonas aeruginosa is a multidrug-resistant bacterial strain with the ability to produce exotoxin A which can pose a serious threat to freshwater ecosystems by having pathogenicity against eukaryotes. Detoxification of exotoxin A and disinfection of P. aeruginosa are the main aims of this study. Using a high dosage of antibiotics might have more toxic effects on ecosystems while cold atmospheric plasma (CAP) can promise reliable, rapid, and environmentally friendly detoxification and disinfection. In this study we produced CAP reinforced by H2O2/H2O cold vapor to detoxify exotoxin A and inactivate P. aeruginosa in freshwater. We used Gammarus roeseli as the indicator of ecotoxicity in freshwater. The mortality of G. roeseli individuals elucidated that 420 s of CAP treatment under a surface dielectric barrier discharge (SDBD) set up can effectively passivize exotoxin A in freshwater by disrupting the protein structure of molecules. Ignorable side effects and changes to the physiochemical properties were observed. On the other hand, 8.2 log reduction of P. aeruginosa viable cells was observed after 300 s of treatment by SDBD. A comparison between the disinfection capacities of SDBD produced CAP and antibiotics revealed that CAP is more effective than most of the common antibiotic agents.