Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization. We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics. Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface. Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide. Finally, the resulting biosurfaces were exposed to antibiotics, i.e. rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively. The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S. epidermidis strain RP12. Furthermore, we examined the in-vitro colonization resistance to S. epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay. The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin). The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model.