Biofilms are collections of microorganisms that attach to a surface and are covered with extracellular matrices produced by these microorganisms from the environment. A biofilm is an ideal place for plasmid exchange, where plasmids can carry genes that regulate resistance to antibiotics so that biofilms play a role in the spread of bacterial resistance to antibiotics. This allegedly due to changes and rearrangement of cell walls so that antibiotics do not easily penetrate it. An alternative method for reducing biofilm S.aureus is photodynamic inactivation. PDI is a method of inactivation of microorganisms that utilize light, photosensitizers, and Reactive Oxygen Species. This present work aims to determine the potential of blue diode laser as an activator of nano doxycycline to reduce Staphylococcus aureus biofilm. The treatment was divided into six groups, the control group without any treatment, the control group with doxycycline photosensitizer, the control group with nano doxycycline photosensitizer, laser treatment group, laser, and doxycycline treatment group, laser, and nano doxycycline treatment groups. The laser treatment group has a variation of exposure time 30s (4.37 J/cm2), 60s (8.73 J/cm2), 90s (13.09 J/cm2), 120s (17.47 J/cm2), and 150s (21.83 J/cm2). Biofilm reduction was measured using an ELISA reader and analyzed using factorial ANOVA. The results showed that 403 nm blue diode laser exposure for 150s with energy density 21.83 J/cm2 could reduce biofilms up to (31.74 ± 1.67)% for the laser treatment group, (65.01 ± 1.67)% for laser and doxycycline treatment groups, (80.25 ± 1.67)% for the laser treatment group and nano doxycycline. So, the exposure of blue diode laser has the potential to activate nano doxycycline to increase the percentage of Staphylococcus aureus biofilm death.