A continuous risk from microbial infections poses a major environmental and public health challenge. As an emerging strategy for inhibiting bacterial infections, plasma-activated water (PAW) has proved to be highly effective, environmental-friendly, and non-drug resistant to a broad range of microorganisms. However, the relatively short lifetime of reactive oxygen and nitrogen species (RONS) and the high spreadability of liquid PAW inevitably limit its real-life applications. In this study, plasma-activated hydrogel (PAH) is developed to act as reactive species carrier that allow good storage and controlled slow-release of RONS to achieve long-term antibacterial effects. Three hydrogel materials, including hydroxyethyl cellulose (HEC), carbomer 940 (Carbomer), and acryloyldimethylammonium taurate/VP copolymer (AVC) are selected, and their antibacterial performances under different plasma activation conditions are investigated. It is shown that the composition of the gels plays the key role in determining their biochemical functions after the plasma activation. The antimicrobial performance of AVC is much better than that of PAW and the other two hydrogels, along with the excellent stability to maintain the antimicrobial activity for more than 14 days. The revealed mechanism of the antibacterial ability of the PAH identifies the unique combination of short-lived species (1O2, ∙OH, ONOO− and O2−) stored in hydrogels. Overall, this study demonstrates the efficacy and reveals the mechanisms of the PAH as an effective and long-term disinfectant capable of delivering and preserving antibacterial chemistries for biomedical applications.