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Nano-sized cobalt oxide particles exhibit unique and fascinating physical, chemical and anti-microbial activity owing to their large surface to volume ratio. The study of metal oxide nanoparticles interaction with pathogenic microorganisms is great importance for various biomedical applications. The main purpose of this work is to study the anti-bacterial and anti-fungal behavior of cobalt oxide (CO3O4) nanoparticles against gram-positive and gram-negative bacterial strains and different fungi. The structure and morphology of CO3O4 nanoparticles were characterized by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and UV-Visible Spectroscopy, respectively. The CO3O4 nanoparticles showed a significant anti-bacterial activity against two gram-positive bacteria, Staphylococcusaureus (G[Formula: see text]), Streptococcus mutans (G[Formula: see text]), and two gram-negative bacteria, Klebsilla pneumonia (G[Formula: see text]), E. coli (G[Formula: see text]), and it shows a good anti-fungal activity against the two different fungal i.e., Aspergillus flavus (F) and Aspergillus niger (F), at different solvents. It was noted that synthesized CO3O4 nanoparticles exhibited the solvent-dependent anti-bacterial activity against all tested bacterial strains. CO3O4 NPs could be employed effectively for anti-microbial applications.