Combining the use of a central composite design (CCD) with a response surface methodology (RSM), Ag-TiO2/Polyurethane (PU) nanocomposites with superior antimicrobial activities and minimal color changes were developed and optimized for self -disinfecting coating. An experimental design was utilized to assess the effects of two independent variables: 1) AgNO3 content and 2) TiO2 content on four measured responses, i.e., the reduction of E. colt and S. aureus, color differences, and the amount of Ag+ release of nanocomposites. The nanocomposites were characterized by Transmission Electron Microscope, and X-ray diffraction. To validate the optimal conditions for real clinical applications for Ramathibodi Hospital (Bangkok, Thailand), the performance evaluations of the coatings such as cytotoxicity test and color differences were conducted, and the antibacterial activities were tested against seven key strains of antibiotic-resistant bacteria. The result suggested that this formulation has high potential for self -disinfecting coating applications.