Infections of intravascular catheters by Staphylococcus aureus is a significant risk factor within the healthcare setting. To treat these infections and attempt salvage of an intravascular catheter, antimicrobial lock solutions (ALSs) are being increasingly used. However, the most effective ALSs against these biofilm mediated infections has yet to be determined and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacy of antibiotics and antiseptics in current clinical use against biofilms produced by reference and clinical isolates of S. aureus. Static and flow biofilm assays were developed using newly described in vivo relevant conditions, to examine the effect of each agent on S. aureus within the biofilm matrix. The antibiotics daptomycin, tigecycline and rifampicin and the antiseptics ethanol and Turalock(TM) inactivated established S. aureus biofilm while other commonly used anti-staphylococcal antibiotics and antiseptic agents were less effective. These findings were confirmed by live/dead staining of S. aureus biofilms formed and treated within a flow cell model. The results from this study demonstrate the most effective clinically used agents and their concentrations which should be used within an ALS to treat S. aureus mediated intravascular catheter related infections.