BACKGROUND: S. aureus biofilm plays a predominant role in the establishment and development of peri-implantitis. It is suggested to combine different modalities as peri-implantitis treatment. The aim of this study was to evaluate the disinfection efficacy of combined application of antiseptics with PDT on Staphylococcus aureus (S. aureus) biofilm formed on titanium(Ti) disks with different surface roughness. METHODS: S. aureus biofilm was incubated on polished and sandblasted large-grit acid-etched (SLA) Ti surfaces for 48 h. 72 contaminated Ti disks (36 polished, 36 SLA) were randomly divided into 6 different groups as follows: (a) PBS, (b) 0.2% chlorhexidine digluconate (CHX), (c) 3% hydrogen peroxide (HO), (d) PDT, (e) 0.2% CHX + PDT, and (f) 3% HO+PDT. Colony forming unit (CFU) was measured to determine antimicrobial effects. Biofilm structure was assessed using scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM). RESULTS: All disinfection methods significantly reduced bacteria amounts compared to control group on both polished and SLA Ti surfaces (P < 0.001). PDT demonstrated stronger decontamination ability in eliminating S. aureus from Ti surfaces than CHX and HO did (P < 0.05). The combined CHX or HO with PDT treatment were more effective in bacterial disinfection than a single administration of these treatments (P < 0.001). CONCLUSION: The combination of CHX or HO administration with PDT was more effective in eradicating S. aureus on both polished and SLA Ti disks in comparison with either treatment alone, suggesting that combined usage of antiseptics with PDT could be a more efficient method for the treatment of peri-implantitis.