Catheter-associated infections can cause severe complications and even death. Effective antimicrobial modification of catheters that can prevent device colonization has the potential of preventing clinical infection. We studied in vitro the antimicrobial activities of central venous catheters impregnated with N-acetylcysteine (NAC), an antibiofilm agent, and a broad-spectrum antibiotic against a range of important clinical pathogens. NAC-levofloxacin-impregnated (NACLEV) catheters were also evaluated for their antiadherence activity. NACLEV catheters produced the most active and durable antimicrobial effect against both Gram-positive and Gram-negative isolates and significantly reduced colonization (P < 0.0001) by all tested pathogens compared to control catheters. These in vitro results suggest that this antimicrobial combination can potentially be used to combat catheter colonization and catheter-associated infection.