The use of bio-processes for synthesizing metal oxide nanoparticles represents a forefront area of research in nanotechnology. This study introduces a rapid and eco-friendly approach for producing cerium oxide nanoparticles (CeO2 NPs) utilizing the aqueous extract of Mollugo oppositifolia L leaves as a catalyst. The synthesized CeO2 NPs underwent comprehensive characterization through scanning electron microscopy (SEM), X-ray diffraction (XRD), ultraviolet–visible spectroscopy (UV–Vis), and Fourier transform infrared spectroscopy (FT-IR). Furthermore, the antimicrobial activity of the CeO2 NPs was assessed in vitro against various bacterial strains, demonstrating a concentration-dependent inhibition zone when exposed to concentrations ranging from 25 to 100 μg/mL. Antitonicity activity results of the synthesized CeO2 NPs revealed significant activity. Further molecular docking studies also revealed that the synthesized CeO2 NPs have better docking ability compared to control Streptomycin in E. coli topoisomerase II DNA gyrase B (PDB ID:1KZN).