Infection of intravascular catheters byStaphylococcus aureus is a significant risk factor within the health care 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 have yet to be determined, and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacies 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 rifampin and the antiseptics ethanol and Taurolock inactivated established S. aureus biofilms, while other commonly used antistaphylococcal 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.T he use of intravascular catheters (IVCs) in modern health care has increased over the last decades. Infection of these devices by surface-adhering bacteria, resulting in catheter-related bloodstream infection (CRBSI), is associated with significant patient morbidity and mortality, prolonged hospitalization, and excess hospital-related costs. The Centers for Disease Control and Prevention (CDC) attributes 12 to 25% mortality among critically ill patients alone to CRBSI (1).Biofilms formed by staphylococci, in particular Staphylococcus epidermidis and Staphylococcus aureus, have for many years been recognized as the most frequent cause of CRBSI (2, 3). These biofilms are highly resistant both to the action of the innate and adaptive immune defense systems and to the action of antimicrobial agents, resulting in persistent infections and treatment failure.The majority of guidelines recommend catheter removal and systemic antimicrobial treatment on suspicion or confirmation of a CRBSI (4). However, clinical circumstances, for example, lack of alternative venous access, bleeding disorders, or comorbid conditions, often preclude device removal. Alternative strategies, such as the use of antimicrobial lock therapy (ALT), to treat these biofilm-related IVC infections, have generated considerable interest in recent years. Antimicrobial lock solutions (ALSs) have been used with variable success to fill the lumen of the IVC in order to eradicate biofilms (5). This technique provides very high concentrations of antimicrobial agents at the site of infection. However, concerns around selection of resistant organisms, toxicity, and treatment failure have thus far limited their widespread application in the ...