Objective:The current study focused on the synthesis of Nickel doped ZnO nanoparticles from the sol-gel technique. Methods: The pre-arranged nanopowder was described by XRD, FTIR, SEM, EDAX, and antibacterial investigations. The X-ray diffraction studies uncover that the incorporated nanoparticles have a wurtzite structure and the molecule size differs from 18 to 31 nm. A change in morphology, after being doped with Nickel, has been noticed. Findings: The Energy Dispersive X-Ray Analysis (EDAX) uncovers that the essential arrangement of arranged examples and the consolidation of the Ni particles into the ZnO cross-section. Escherichia coli, Staphylococcus aureus, Aspergillus flavus, and A. niger were used to test the antibacterial activity of Nidoped ZnO nanoparticles using the plate dispersion method. Novelty: The antibacterial activity improvement with Ni doping effect is attributed to the enhancement of electronic defects including vacancy defect Ni and interstitial defect ZnO with Ni doping Concentration. These defects behave as electrons and holes source necessary for reactive oxygen species (ROS) responsible for bacteria's destruction.