Millions of people die every year as a result of infections’ multidrug resistance (MDR) pathogens. Therefore, the creation of novel antibiotics is urgently required for the treatment of bacterial illnesses that are resistant to a variety of drugs. The current study concentrated on the development of copper oxide nanoparticles conjugated with Vitamin-E (CuO NPs-Vit E complex), which was evaluated using various approaches like FTIR (Fourier transform infrared spectroscopy), UV-Vis (ultraviolet-visible spectroscopy), SEM (scanning electron microscopy), XRD (X-ray diffraction). The antibacterial and anti-biofilm activity was investigated against MDR Escherichia coli and Staphylococcus aureus. The antioxidant properties of CuO NPs-Vit E complex were determined using DPPH and FRAP assay. The CuO NPs-Vit E complex showed excellent anti-bacterial, anti-biofilm, and antioxidant activities. Moreover, it was found to be biocompatible and non-toxic to normal cells. Therefore, the synthesized CuO NPs-Vit E complex can be used for the creation of new drugs for the treatment of multidrug-resistant bacterial infections.