This study presents the fabrication and characterization of novel bactericidal Nanoceria doped composite nanofibers. Nanoceria doped composite nanofibers fabricated by physically blending Polyurethane (PU) with two biopolymers such as Cellulose acetate (CA) and Zein. The Nanoceria were prepared by electrospinning of a sol–gel composed of cerium nitrate hexahydrate, and polyvinylpyrrolidone and followed by calcination and sonication. The physicochemical characterizations of the synthesized Nanoceria doped composite nanofibers were carried out by X-ray diffraction pattern, field emission scanning electron microscopy, and transmission electron microscopy. The antibacterial activity was tested against most common pathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae, Salmonella enterica (Gram-negative), Staphylococcus aureus and Enterococcus faecalis (Gram-positive). The Nanoceria doped composite nanofibers have demonstrated effective toxicity against both the Gram positive and Gram negative bacterial strains. The obtained results indicate that the inhibition is dependent on the concentration of Nanoceria doped composite nanofibers. TEM analysis reveals that Nanoceria caused disruption of cell membranes which led to the irreversible damage to the cell envelope eventually leading to cell death. Generally, these Nanoceria doped composite nanofibers were depicted as a promising nanomaterial for exceptional antibacterial treatment and can be used as another smart material for biomedical and industrial applications.