A viable hydrothermal technique has been explored for the synthesis of copper doped Zinc oxide nanoparticles (Cu-doped ZnO-NPs) based on the precursor's mixture of Copper-II chloride dihydrate (CuCl2.2H2O), Zinc chloride (ZnCl2), and potassium hydroxide (KOH). X-ray diffraction (XRD) reported the hexagonal wurtzite structure of the synthesized Cu-doped ZnO-NPs. The surface morphology is checked via field emission scanning electron microscopy (FE-SEM), whereas, the elemental compositions of the samples were confirmed by Raman, and X-ray photoelectron spectroscopy (XPS), respectively. The as-obtained ZnO-NPs and Cu-doped ZnO-NPs were then tested for their antibacterial activity against clinical isolates of Gram-positive (Staphylococcus aureus, Streptococcus pyogenes) and Gram-negative (Escherichia coli, Klebsiella pneumonia) bacteria via agar well diffusion method. The zone of inhibition (ZOI) for Cu-doped ZnO-NPs was found to be 24 and 19 mm against S. Aureus and S. pyogenes, and 18 and 11 mm against E. coli and K. pneumoniae, respectively. The synthesized Cu-doped ZnO-NPs can thus be found as a potential nano antibiotic against Gram-positive multi-drug resistant bacterial strains.