The relative resistance of S. epidermidis implant-associated infections to antibiotic therapy has been ascribed to a protective function of the gluelike biofilm matrix produced by strains of S. epidermidis in contact with artificial surfaces. Using a standardized S. epidermidis biofilm assay we determined the periods of exposure required by various antibiotics to produce cessation of biofilm metabolic activity. Rifampin has the superior rate of action, producing substantial disruption of biofilm activity by 7 hr of exposure, but leading to replacement of the susceptible bacterial cells by rifampin-resistant mutant survivors. Other antibiotics required longer periods of exposure, in excess of 48 hr, but produced a bactericidal outcome. Combinations of antibiotics with rifampin produced strikingly divergent results. Cefazolin and vancomycin (cell wall active antibiotics) produced a bactericidal outcome at 16 hr of exposure, whereas gentamicin (aminoglycoside) neutralized the rapid action of rifampin with metabolic activity maintained at 48 hr. We confirmed the selectively protective function of the S. epidermidis biofilm with regard to antibiotic action. In vitro biofilm assays may be of value in guiding antibiotic therapy in S. epidermidis implant-associated infection.