In this research, Cu/Cr codoped ZnO nanoparticles (Zn₀.₉₉₋ₓCu₀.₀₁CrxO) were prepared using the sol-gel technique. The dopant ratio was systematically varied, ranging from x = 0.01 to 0.05 in increments of 0.01. This variation allowed for an exploration of the impact of defects on the structural, microstructural, optical, and antibacterial properties of Cu/Cr-codoped ZnO nanoparticles. Structural analysis of the Zn₀.₉₉₋ₓCu₀.₀₁CrxO nanoparticles was conducted using X-ray diffraction. The findings confirmed the presence of a hexagonal wurtzite structure in the ZnCuCrO nanoparticles, as determined by the c/a ratios in the range of 1.6013 and 1.6039. The surface morphology, crystallite size, and nanoparticle shapes were determined through scanning electron microscopy. The nanoparticle elemental compositions were analyzed through electron dispersive spectroscopy. The optical characteristics of the samples were assessed using a UV spectrophotometer. The energy band gaps for the nanoparticles were computed, and we discussed how dopant elements and defects affected their optical properties. We determined the refractive index using five distinct models. Notably, the highest band gap was observed for Zn₀.₉4Cu₀.₀₁Cr₀.₀5O (Eg=3.27 eV).Positron annihilation lifetime spectroscopy was employed to investigate the defect type, defect density, and crystal quality of Cu/Cr-codoped ZnO nanoparticles. The shortest lifetime τ1 obtained the highest value (0.181 ns) for Zn0.97 Cu0.01Cr0.02O nanoparticles. Zn₀.₉₉₋ₓCu₀.₀₁CrxO NPs were assessed for their antibacterial effects on E. coli (gram-negative) and S. aureus (gram-positive). Cu/Cr codoped ZnO nanoparticles show potential as materials for optoelectronic and sensor applications due to their broad band gap (Eg > 3) and high refractive index (n > 2).