Antibacterial resistance requires an advanced strategy to increase the efficacy of current therapeutics in addition to the synthesis of new generations of antibiotics. In this study, copper oxide nanoparticles (CuO-NPs) were green synthesized using Moringa oleifera root extract. CuO-NPs fabricated into a form of aspartic acid-ciprofloxacin-polyethylene glycol coated copper oxide-nanotherapeutics (CIP-PEG-CuO) to improve the antibacterial activity of NPs and the efficacy of the drug with controlled cytotoxicity. These NPs were charachterized by Fourier transform infrared spectroscopy (FTIR), x-rays diffraction spectroscopy (XRD), scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS). Antibacterial screening and bacterial chemotaxis investigations demonstrated that CIP-PEG-CuO NPs show enhanced antibacterial potential against Gram-positive and Gram-negative clinically isolated pathogenic bacterial strains as compared to CuO-NPs. In ex-vivo cytotoxicity CIP-PEG-CuO-nano-formulates revealed 88% viability of Baby Hamster Kidney 21 cell lines and 90% RBCs remained intact with nano-formulations during hemolysis assay. An in-vivo studies on animal models show that Staphylococcus aureus were eradicated by this newly developed formulate from the infected skin and showed wound-healing properties. By using specially designed nanoparticles that are engineered to precisely transport antimicrobial agents, these efficient nano-drug delivery systems can target localized infections, ensure targeted delivery, enhance efficacy through increased drug penetration through physical barriers, and reduce systemic side effects for more effective treatment.