The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, R12-AgNPs were produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature. The biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyse the decomposition of H2O2 to generate hydroxyl radical. After combination of R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was observed for both E. coli and S. aureus, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.