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This paper reports the structural, optical and antimicrobial study of Ce_1−xFe_xO_2−δ (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mn>0</mn><mo>≤</mo></mrow></semantics></math></inline-formula> x <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>≤</mo><mn>20</mn></mrow></semantics></math></inline-formula>) nanoparticles (NPs) synthesized using a microwave-assisted hydrothermal method. The XRD pattern analysed using Rietveld refinement method clearly infers that all the samples exhibit single phase nature and exclude the possibility of an impurity phase. The lattice parameters and unit cell volume were found to decrease with an increase in Fe-doping content in CeO₂ nanoparticles. The crystalline size determined using XRD pattern and TEM micrographs was found to decrease with Fe doping in CeO₂. Selective area electron diffraction (SAED) pattern also demonstrated the crystalline nature of the Fe-CeO₂ nanoparticles. Optical properties studied using UV–vis spectroscopy indicated that band gap decreased with an increase in Fe doping. The electrical properties have been investigated via dielectric constant, dielectric loss and AC conductivity. The dielectric constant was found to increase in the Fe-doped CeO₂ nanoparticles, while AC conductivity was found to be reduced, which shows good dielectric behaviour of the Fe-doped CeO₂ nanoparticles. The antibacterial activity of the synthesized NPs was achieved under ambient conditions with different bacteria, and the results showed that the properties were different for both bacteria. The antimicrobial activity reflects the possibility to develop Fe-doped CeO₂ NPs as antibacterial agents against extensive microorganisms to control and prevent the spread and persistence of bacterial infections.