Slime-producing staphylococci frequently colonize catheters, and when they are embedded in biofilm, they become resistant to various antibiotics. In the study that is described, the comparative efficacies of vancomycin, clindamycin, novobiocin, and minocycline, alone or in combination with rifampin, were tested in an in vitro model of colonization. The model consisted of the modified Robbins device with antibiotic-impregnated cement filling the lumen of catheter segments. The synergistic combination of minocycline and rifampin was the most efficacious in preventing bacterial colonization of slime-producing strains of Staphylococcus epidermidis and Staphylococcus aureus to catheter surfaces. A similar trend was observed when the inhibitory activities of polyurethane catheters coated with minocycline and rifampin were compared with the inhibitory activities of catheters coated with other antimicrobial agents. The inhibitory activities of catheters coated with minocycline and rifampin against S. epidermidis, S. aureus, and Enterococcus faecalis strains, for example, were significantly better than those of catheters coated with vancomycin (P < 0.05). The inhibitory activities of catheters coated with minocycline and rifampin against gram-negative bacilli and Candida albicans were comparable to those of catheters coated with ceftazidime and amphotericin B, respectively. We found that the combination of minocycline and rifampin is unique and highly effective in preventing the colonization of catheters with slime-producing staphylococci and that it also displays a broad-spectrum inhibitory activity against gram-negative bacteria and yeast cells.