Calcium oxide (CaO) nanoparticles have gained wide attention in various environmental applications, including water treatment and microbial pollution control. This research provides novelty in innovating new strategies in water management and waste treatment based on advanced technology with a nanoscience approach by utilizing local resources.This study aims to synthesize CaO nanoparticles using a green chemistry method using a bioreductor from Bitti (Vitex cofassus) plant extract. This green chemistry approach is not only environmentally friendly but also efficient in producing stable nanoparticles with controlled size. Nanoparticle characterization was performed using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) to determine their crystal structure, morphology, and particle size. The results showed that calcium oxide nanoparticles have FCC (Face-centered cubic) crystal phase, uneven surface morphology, and spherical shape and have an average particle size of 24.87 nm. The antibacterial activity of calcium oxide nanoparticles on Escherichia coli test bacteria with variations in nanoparticle concentrations (1, 3, and 5%) each had an average inhibition zone diameter of 9.59; 10.78; and 11.78 mm, positive control (Chloramphenicol) of 12.65 mm, and negative control (sterile aqua) of 0 mm, while in Staphylococcus aureus test bacteria with nanoparticle concentration variations (1, 3, and 5%) each had an average inhibition zone diameter of 10.26; 11.15; and 14.15 mm, positive control (Chloramphenicol) of 12.82 mm, and negative control (sterile aqua) of 0 mm. CaO nanoparticles have greater effectiveness on Staphylococcus Aureus bacteria than Escheria Coli with the ability to inhibit and kill both types of bacteria. The application of these nanoparticles as antibacterial agents can effectively reduce microorganism waste in the aquatic environment, so they have the potential to be used as a solution for environmentally friendly microbial waste treatment.