In this work, flower shape cerium oxide (CeO2) nanosheets were synthesized at room temperature through a facile precipitation method without adding any surfactant. The synthesized nanosheets were characterized by scanning electron microscope (SEM), high resolution transmission electron microscope (HRTEM), Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffractometry (XRD) and X‐ray photoelectron spectroscopy (XPS). Results showed that the CeO2 nanosheets form a petal‐like morphology and have fluorite crystal structure with no crystal phase impurity while the average crystallite size of nanosheets i. e. ~10 nm. The CeO2 nanosheets are applied as an antimicrobial agent and the antibacterial property against G+ and G− bacteria i. e. (E. coli and S. Aureus) were studied. The obtained results showed that the synthesized CeO2 nanosheets had good antibacterial performance at different concentrations, which should be attributed to the reversible Ce3+/Ce4+ valence states and greater oxygen vacancies. XPS study of CeO2 nanosheets after antibacterial test confirmed the existence of Ce3+ ions and oxygen vacancies. CeO2 nanosheets have showed the greater potential as antibacterial nanomaterials against both the bactericides.