A vascular catheter may be saved if an intraluminal antibiotic lock treatment is used in the case of catheter-related bloodstream infections. These recently developed antibiotics were compared to the older antibiotics vancomycin and minocycline against methicillin-resistant Staphylococcus aureus (MRSA) embedded in biofilms in this in vitro study. We examined the emergence of MRSA after four hours of daily catheter lock therapy that was resistant to these antibiotics alone and with rifampin. Compared to linezolid, vancomycin, and the negative control, minocycline, daptomycin, and tigecycline are more effective at inhibiting MRSA in biofilms after the first day of exposure (P 0.001), with minocycline being the most active and daptomycin and tigecycline having weak activity, similar to the negative control. Among the antibiotics tested, daptomycin was the fastest in eliminating MRSA from biofilm after three days of exposure for 4-hour periods, followed by minocycline and tigecycline, which were faster than linezolid, rifampin, and vancomycin (P 0.001). After five days of exposure to MRSA continuously for four hours each day, rifampin alone was least effective at eradicating MRSA from biofilms because it promoted rifampin-resistant MRSA growth. It has been found that when rifampin is combined with additional antibiotics, the combination is far more effective at eliminating MRSA colonization than any single antibiotic alone. Daptomycin, minocycline, and tigecycline lock therapy also needs to be researched, as should the potential for rifampin's antistaphylococcal activity as a single agent, but not as a combined solution.