Excessive use of antibiotics across the globe has raised the concerns of antibiotic-resistant pathogenic microbial strains. Such strains threaten our ability to treat common infections because they are difficult to treat thus enhance the risk of disease spread, severe illness and death. Thus it increases the demand to identify alternatives to those traditional antibacterial medicines in order to fight against antibiotic-resistant microorganisms. In this concern, among new strategies, nanomaterials as antibacterial have shown tremendous success because they are biocompatible, show broad-spectrum bactericidal activity, distinct functioning mechanism towards pathogenic bacteria, and negligible toxicity towards healthy cells. Recently, cerium oxide nanoparticles (CeO₂ NPs) have shown excellent pro-oxidant activity due to their unique characteristics of mixed valance states. The reversible conversion between the two oxidation states is an auto-regenerative cycle (Ce⁴⁺→Ce³⁺→Ce⁴⁺) operating at the surface of NPs. Frequently, the nanozymes have weak enzymatic activity, and therefore, strategies are required to boost the activity. Herein, we show that CeO₂ NPs exhibiting oxidase enzyme-like activity, and thus generate superoxide radicals at acidic pH but not at neutral pH. Interestingly, the display of oxidase-like activity could be shifted to neutral pH with the use of boosting agent. Therefore, the screening of nucleotides (such as Adenosine, AMP, ADP, ATP, CTP, GTP, UTP, NADH, FAD) was carried out as an external boosting agent to aid CeO₂ NPs in generating superoxide radicals at neutral pH. Among all nucleotides, ATP was found as the best boosting agent to promote the oxidase enzyme-like activity of CeO₂ NPs. The superoxide radical generation, during the oxidase-mimetic activity of CeO₂ NPs with ATP, was confirmed using dihydroethidium and p-benzoquinone while indole propionic acid, ethanol, and terephthalic acid ruled out the formation of hydroxyl radicals. Utilizing the superoxide radical generation ability of CeO₂ NPs, the anti-bacterial activity against Staphylococcus aureus and Escherichia coli was tested using different assays. Quantitative method revealed ~70% and ~80% cell death (S. aureus) and ~40% and ~60% cell death (E. coli) after the exposure of CeO₂ NPs + ATP for 3 hrs and 6 hrs respectively. Scanning electron microscopy study revealed the important changes in the morphology of the exposed bacterial population. Distortion of tetrad arrangement, loss of cytoplasmic content (S. aureus), cell membrane damage and pore formation in cell wall (E. coli) were the major causes for the bacterial cell death. Time-dependent Live/Dead estimation assay also revealed the highest bacterial cell death exposed to CeO₂NPs + ATP after the treatment of 6 hrs. Hence, inclusively it was observed that the combination effect of CeO₂ NPs + ATP was more successful in generating superoxide radicals as compared to the CeO₂ NPs alone and exhibit antibacterial activity. Since, this strategy does not involve the use of antibiotics, it is concluded that this approach could be a novel and an efficient alternative to kill the antibiotic-resistant pathogens.