Infection is the entry of bacteria or pathogenic microorganisms into the body that can cause pain. For example, Staphylococcus aureus infection is caused by direct contamination of wounds, either post-surgical or infection after trauma. This study aims to determine the effectiveness of nano doxycycline as an exogenous photosensitizer agent in antimicrobial photodynamic inactivation (PDI) by using laser diode activator (403±0.02) nm against Staphylococcus aureus bacteria. The study was divided into three groups which were T0 as the untreated control group, T1 as treatment group with nano doxycyclin but without laser exposure, T2 as Treatment group with nano doxycyclin and laser treatments at various exposure times (30, 60, 90, 120, 150) seconds. This study used the Total Plate Count (TPC) method to find out the percentage decrease in the number of colonies. The results of statistical analysis showed that there were significant differences in the number of bacterial colonies due to the addition of nano doxycycline 0.1% and laser diode exposure (403.00±0.02) nm at (150.00±0.01) second with a energy density of (21.83±0.02) J/cm2. The results showed the addition of nano doxycycline was effective to reduce the number of bacterial colonies with a percentage of deaths of (85.34±1.20)%. So, antimicrobial photodynamic inactivation using laser diode with nano doxycyclin is effective to reduce the number of S. aureus bacteria.