In this modern era, antibiotic resistance is a significant issue that poses a threat to public health. Nanotechnology is an emerging field of science because nanoparticles could be the best alternative to antibiotics. Most nanoparticles are prepared by the green synthesis method because of their less toxicity, low cost, and non-hazardous nature. In this study, cobalt oxide nanoparticles (Co3O4-NPs) were synthesized from roots extract of Ziziphus Oxyphylla Edgew by using cobalt chloride hexahydrate. After the successful synthesis of nanoparticles, various methods were used to analyze these nanoparticles, including Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction analysis, and energy dispersive analysis of x-ray. Scanning electron microscopy images reveal the spherical and irregular structure of Co3O4-NPs shaving a particle size between 40 to 60 nm. X-ray diffraction analysis revealed the crystalline nature of cobalt oxide nanoparticles with face-centered cubic structure and a size of 15-20 nm. The antibacterial activity of Co3O4-NPs was analyzed for different dilutions against two different bacteria: gram-negative (E. coli) and gram-positive (S. aureus) bacteria. The maximum zone of inhibition against gram-negative E. coli was calculated as 23.1 mm and 14.8 mm against S. aureus at a dilution of 16 mg/ml of cobalt oxide nanoparticle. This revealed the wide spectrum of antibacterial activity of the synthesized nanoparticle. It is suggested that root extract of cobalt oxide nanoparticles could be of great importance in pharmaceutical and medical science for their antimicrobial activity.