Copper and nickel nanoparticles are known to be one of the multifunctional inorganic nanoparticles with effective antibacterial activity. This study aims to determine the antimicrobial efficacy of chemical synthesized copper and nickel nanoparticle against various bacterial and fungal pathogens. Grinding method is used to synthesize copper and nickel nanoparticles. In this paper,the new cationicthiol polyurethane surfactants with different alkyl chain length were synthesized (PQ8, PQ10 and PQ12). The chemical structure of the synthesized surfactants was confirmed using infra-red spectroscopy (IR) and proton nuclear magnetic resonance spectroscopy (+H NMR). The nanostructure of the synthesized surfactant with copper and nickel nanoparticles with diameters ranging from 10 to 55 nm was prepared and characterized using ultra violet spectrophotometer (UV),infra-red spectroscopy (IR) and transmission electron microscope (TEM). The results declare formation and stabilization of copper and nickel nanoparticle using synthesized cationic surfactants. Antimicrobial activity of the synthesized cationic surfactants and their nanostructure with copper and nickel nanoparticles were evaluated against pathogenic bacteria and fungi. The antimicrobial activity showed the enhancement in the antimicrobial activity of the synthesized cationic surfactants in the nanostructures form.