Hydrogen peroxide, povidone-iodine, and chlorhexidine are antiseptics that are commonly added to irrigants to either prevent or treat infection. There are little clinical data available that demonstrate efficacy of adding antiseptics to irrigants in the treatment of periprosthetic joint infection after biofilm establishment. The objective of the study was to assess the bactericidal activity of the antiseptics onS. aureusplanktonic and biofilm. For planktonic irrigation,S. aureuswas exposed to different concentrations of antiseptics.S. aureusbiofilm was developed by submerging a Kirschner wire into normalized bacteria and allowing it to grow for forty-eight hours. The Kirschner wire was then treated with irrigation solutions and plated for CFU analysis. Hydrogen peroxide, povidone-iodine, and chlorhexidine were bactericidal against planktonic bacteria with over a 3 log reduction (p< 0.0001). Unlike cefazolin, the antiseptics were not bactericidal (less than 3 log reduction) against biofilm bacteria but did have a statistical reduction in biofilm as compared to the initial time point (p< 0.0001). As compared to cefazolin treatment alone, the addition of hydrogen peroxide or povidone-iodine to cefazolin treatment only additionally reduced the biofilm burden by less than 1 log. The antiseptics demonstrated bactericidal properties with planktonicS. aureus; however, when used to irrigateS. aureusbiofilms, these antiseptics were unable to decrease biofilm mass below a 3 log reduction, suggesting thatS. aureusbiofilm has a tolerance to antiseptics. This information should be considered when considering antibiotic tolerance in establishedS. aureusbiofilm treatment.