At present, microbes have enormous potential to become a major global public health issue. For example, Escherichia coli is the prominent cause of cholecystitis, urinary tract infections, and other infections. Due to its outstanding antibacterial properties, nitric oxide (NO) is essential for biological processes. Additionally, enzymatic hydrolysis using polyethylene terephthalate hydrolase (PETase) is one of the promising methods for PET upcycling. First, recombinant PETase was used to enzymatically treat waste PET fibers, and polyethylenimine (PEI) was added as a secondary amine donor. Subsequently, the aminated PET fiber was inserted into a reactor charged with NO gas (10 atm, 3 days) to obtain N-diazeniumdiolate (NONOate) products that can inhibit bacteria growth. In this study, the first strategy for antibacterial applications by NO-releasing PETase-hydrolyzed PET fibers was demonstrated. NO-conjugated PET fibers were successfully prepared which exhibits a continuous NO release profile over 12 h. The surface properties of functionalized PET fibers were successfully confirmed by fourier-transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and Griess assay. The antibacterial test indicated a reduction of Escherichia coli by 90.2% and Staphylococcus aureus by 71.1% after exposure to the functionalized material. Therefore, this novel antibacterial agent may offer great potential applications in the medical field.