This study synthesized Zn1-xCuxO nanoparticles (x=0.0, 0.02, and 0.04) from Calotropis gigantea leaf extract utilizing green synthesis. The effect of Copper (Cu) doping on optical properties, structure, and morphology were examined using XRD, SEM, and UV-Vis spectroscopy. XRD confirmed hexagonal wurtzite unit cell. As Cu concentrations increased, nanoparticle micro-strain and dislocation density decreased while bond length, crystallite size, lattice constant, and unit cell volume increased. The atomic packing percentage was 73.12%, and the bond length ranged from 1.988 to 1.990 Å. FESEM revealed spherical nanoparticles. Doping red-shifts the band gap of nanoparticles from 3.31 to 3.02 eV. The green synthesized Zn1-xCuxO nanoparticles are used for the first time as a nano antibiotic. The Zn1-xCuxO nanoparticles were tested for antibacterial activity against gram-positive (S. pyogenes) and gram-negative (E. coli) using the agar well diffusion method. The antibacterial results demonstrated that Cu-doped ZnO NPs had a larger inhibitory zone diameter throughout the chosen bacteria.