In this study, a CuSO4.5H2O solution was reduced with mango extract at the optimal pH of 11 to create copper oxide (CuO) nanoparticles in a quick, simple, and ecologically friendly manner. CuO nanoparticle formation was indicated by the color changes from blue to light green, then to greenish black, and lastly to green precipitate after a few days. The structure of the biosynthesized nanoparticles was described using ultraviolet-visible (UV-Vis) and Fourier transform infrared (FT-IR) spectroscopy. The purity of the crystal structure was also investigated using X-ray diffraction (XRD).UV-Vis spectroscopy was used to monitor and control the reduction process, which was simple. The results showed that the electron's surface Plasmon resonance caused the maximum absorption to appear at 286 nm, which is significantly different from the maximum absorption formed by the pure mango leaf extract at 260 nm.This has clearly revealed the formation of CuO nanoparticles. For this reason, the presence of Qualitative screening techniques (looking for color changes) were used to identify the active flavonoids, phenols, tannins, alkaloids, saponins, glycosides, and carbohydrates that were present in the biomass of the mango extract both before and after reduction. The antibacterial efficacy of these biologically produced CuO nanoparticles was evaluated against four human pathogens: Kleibsiella pneumonia, Escherichia coli, Streptococci pyrogens, and Staphylococcus aurous. With a maximum zone of inhibition of 13 mm, they were discovered to have a considerable impact on regulating the proliferation of human infections. As a result, the nonmaterial can be used in various fields for antibacterial purposes when mango is used to synthesize CuO nanoparticles