The clinical manifestations of infection by bacteria forming biofilms is their resistance to antibiotic treatment. So, we need an alternative method that is both effective and selective for killing of Staphylococcus aureus (S. aureus) biofilm bacteria. One of which is a method of photodynamic (PDT) by utilizing light and photosensitizer. The purpose of this study is to build and determine the efficacy of CNC-laser diode, combine with/without chlorophyll to eliminate the microbial S. aureus biofilm. CNC (Computer Numerical Control) laser instrument consisted of light source (diode laser), sample holder and controller to control of the intensity values, the number of microplate lines, time duration of irradiation, the stepper motor rotation according to predetermined coordinates, and control the fan. Fan control is aimedto maintain inside temperature of the sample box at room temperature. The characterization of CNC-laser showed the stability of laser power and temperature during irradiation. The 405 nm laser treatment group combine with and without chlorophylls resulted in statistically non-significant efficacy of log CFU (p<0.05) compared to each other. Best efficacy was shown under laser irradiation time of 75 seconds with endogenous PS with the efficacy of 18% and chlorophylls of 22.28%. The result of biofilm viewing of confocal microscopy shows that CNC-laser treatment with exogenous PS (chlorophylls) could eliminate the S. aureus biofilm. So CNC-diode laser combine with chlorophylls can eliminate Staphylococcus biofilm.