The emergence of drug-resistant bacterial strains has challenged the use of traditional antibiotic therapies. Therefore, there is an urgent need to develop novel antibacterial materials or methods to effectively eradicate bacteria. Herein, we demonstrated a novel antibacterial system consisting of cupric oxide nanoparticles combined with ascorbic acid (CuO NPs/AA), which could act as a potent nanoantibiotic targeting both Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) in vivo and in vitro, without noticeable cytotoxicity to mammalian tissue. The CuO NP/AA antibacterial system killed these microbes through a combined mechanism involving biofilm inhibition, cell membrane destruction, DNA damage, and enzyme inactivation. The antibacterial properties of CuO NPs/AA were mainly attributed to the generation of reactive oxygen species (e.g., ·OH) produced through the intrinsic ascorbate oxidase- and peroxidase-like activities of CuO NPs, highlighting their use as antibacterial nanozymes to treat bacterial infections.