Cerium oxide nanoparticles (CONPs) were prepared using ultrasound assisted leaf extract of Prosopis juliflora acting as a reducing as well as stabilizing agent. The synthesized CONPs were characterized by ultraviolet-visible absorption spectroscopy (UV-Vis), particle size analyzer (PSA), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electron microscopy (HRTEM). From the UV-Vis analysis, the optical band gap of the prepared CONPs (Eg = 3.62 eV) was slightly increased as compared to the bulk ceria (Eg = 3.19 eV). The phytochemicals in the extract reduced the particle size to 3.7 nm ± 0.3 nm, as it is evident from the PSA. FT-IR results confirmed the Ce-O stretching bands by showing the peaks at 452 cm-1. The Raman spectrumshowed a characteristic peak shift for CONPs at 461.2 cm-1. XRD analysis revealed the cubic fluorite structure of the synthesizednanoparticles with the lattice constant, a of 5.415 Å and unit cell volume, V of 158.813 Å3. XPS signals were used to determine the concentration of Ce3+ and Ce4+ in the prepared CONPs and it was found that major amount of cerium exist in the Ce4+ state. HRTEM images showed spherical shaped particles with an average size of 15 nm. Furthermore, the antibacterial activity of the prepared CONPs was evaluated and their efficacies were compared with the conventional antibiotics using disc diffusion assay against a set of Gram positive (G+) bacteria (Staphylococcus aureus, Streptococcus pneumonia) and Gram negative (G-) bacteria (Pseudomonas aeruginosa, Proteus vulgaris). The results suggested that CONPs showed antibacterial activity with significant variations due to the differences in the membrane structure and cell wall composition among the two groups tested.