Background: The surge in bacteremia and healthcare-associated infections, driven by aging populations and antimicrobial resistance, prompted research into nanotechnology. Copper Oxide Nanoparticles (CuONPs) have emerged as a promising candidate due to their potent antibacterial properties. Muntingia calabura, renowned for its medicinal uses, presents a novel avenue for CuONP synthesis. This study aimed to evaluate the antibacterial efficacy of CuONPs against Staphylococcus aureus and Escherichia coli. Materials and Methods: M. calabura leaves extracts were utilized to synthesize CuONPs from Copper (II) Sulfate Pentahydrate. CuONPs morphology was characterized by Scanning Electron Microscopy. Subsequently, the antibacterial properties of the CuONPs with various concentrations of 25%, 50%, 75% and 100% were analyzed against S. aureus and E. coli using agar well diffusion and MIC determination vis-à-vis gentamicin as the control. Results: The results showed antibacterial potential with 75% leaf extract concentration showing the highest Zone of Inhibition (ZOI) and 50% as Minimum Inhibitory Concentration (MIC) for S. aureus. While, 50% of the concentration showed the highest ZOI and MIC at 25% for E. coli. ANOVA and Tukey’s post-hoc determined a significant difference between the different concentrations of the CuONPs (p-values=0.000 and 0.035). However, only the antibacterial activity of the CuONPs against E. coli showed no significant difference with the positive control (p-value=0.125). Conclusion: Although the synthesized CuONPs are deficient compared to positive control gentamicin, it manifests significant antibacterial potential against S. aureus and E. coli and is recognizable as a potential alternative in combating antibacterial resistance.