The construction of plant-based nanoparticles (NPs) has several benefits over traditional physico-chemical techniques and useful in both biology and medicinal field. This study compared the synthesis of CuO nanoparticles (CuO NPs) using chemical methods (using polymers such as PEG and PVP) and from leaf extract using medicinal plant Eucalyptus globoulus (E. globulus) by green approaches. Using a simple precipitation process, cupric nitrate served as a precursor for the synthesis of CuO NPs. X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Scanning electron microscopy (SEM) and Ultraviolet-visible spectrophotometer (UV-Vis) were used to examine the structural and optical characteristics of NPs. When compared to conventional medications, the E. globulus mediated CuO NPs (Eu-CuO NPs) demonstrated remarkable antibacterial action against clinical infections, indicating that plant-based NPs synthesis can be an immense way to create biomedical products that are both adaptable and environmentally friendly. Antibacterial studies of NPs tested against Escherichia coli (E. coli) and antifungal activities were treated against Aspergillus niger (A. niger), Aspergillus flavus (A. flavus). Research on antibacterial and antifungal properties revealed that Eu-CuO NPs prepared through green approach photo dynamically inactivated more efficiently than those prepared from chemical approach.