Microbial mediated synthesis of metallic nanoparticles constitutes as effective and promising approach for the development of antibacterial materials in the field of bioengineering and biomedicine. We prepared Cadmium oxide nanoaprticles (CdO NPs) utilizing Penicillium oxalicum, and cadmium acetate solution via coprecipitate method. The elemental composition and morphology of these synthesized CdO NPs were examined through X-ray diffraction (XRD), UV-Vis absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), and Energy dispersive spectroscopy (EDS). Furthermore, we evaluated the bactericidal potential of prepared CdO NPs using Escherichia coli (E.coli), Staphylococcus aureus (S.aureus), Bacillus cereus (B.cereus), and Pseudomonas aeruginosa (P. aeruginosa). Dimethyl sulfoxide was used as negative control while erythromycin was used as positive control. The XRD spectrum revealed cubic crystalline nanoparticles with 22.94 nm size and UV showed absorbance peak at 297 nm with 2.5 eV band gap energy. FTIR depicted O─H and carboxylic groups along with CdO stretching vibration. EDS showed the presence of organic compounds on Cd and O over NPs surface. SEM results revealed the spherical shape of the CdO NPs. The synthesized NPs exhibited highly potent bactericidal activity against selected strains and demonstrated less optical density of 0.086 after 24 hr. Owing to the significant antibacterial activity of CdO NPs, the broad application prospects of these nanoparticles CdO NPs in extensive biomedical applications is indicated.