Copper nanoparticles were synthesized using modified polyol method by the reduction of copper acetate hydrate in the presence of Tween 80 by refluxing between 190° and 200°C. The X-ray diffraction pattern was used to analyze the formations of phase and crystal structure. The antimicrobial activity was carried out against Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, fungus like Aspergillus flavus, Aspergillus niger and Candida albicans. The copper nanoparticles showed more inhibitory activity in bacteria than the fungus and it also showed more zone of inhibition in E.coli (26mm) than C. albicans (23mm).