Infection associated with medical devices may involve bacterial biofilms, a possible cause of antibiotic resistance. Protamine, a basic polypeptide, depresses the metabolic activity of Staphylococcus epidermidis in a standardized biofilm assay. The resistance of the biofilm preparations to many antibiotics, with the sole exception of rifampin, was confirmed. Rifampin produced predominant lysis and killing with foci of genetically programmed resistance. The combination of protamine with antibiotics produced no change, except the combination with rifampin where clear classic synergism with a totally bactericidal outcome was demonstrated. Protamine is a member of the larger family of charged polycations, some of which possess membrane potential altering properties, possibly interfering with the protective nature exhibited by the negatively charged bacterial biofilm matrix.