To compare the efficacy of five antibiotics against methicillin-susceptible Staphylococcus aureus (MSSA) biofilms on the surface of four vascular grafts. In vitro study of two clinical MSSA strains (MSSA2 and MSSA6) and four vascular grafts (Dacron, Dacron-silver-triclosan, Omniflow-II, and bovine pericardium). After a 24-hour incubation period, the graft samples were divided into six groups: growth control (no treatment), ciprofloxacin 4.5mg/L, cloxacillin 100 mg/L, dalbavancin 300 mg/L, daptomycin 140 mg/L, and linezolid 20 mg/L. Quantitative cultures were obtained and results expressed as log10 colony-forming units per milliliter (CFU/mL). Analysis of variance was performed to compare biofilm formation between the different groups. Mean ± SD MSSA2 count on the growth control Dacron graft was 10.05 ± 0.31 CFU/mL. Antibiotic treatment achieved a mean reduction of 45%; ciprofloxacin was the most effective antibiotic (64%). Baseline MSSA2 counts were very low on the Dacron-silver-triclosan (0.50 ± 1.03 CFU/mL) and Omniflow-II (0.33 ± 0.78 CFU/mL) grafts. On the bovine pericardium patch, the count was 9.87 ± 0.50 CFU/mL, but this was reduced by a mean of 45% after antibiotic treatment (61% for ciprofloxacin). Mean MSSA6 count on the growth control Dacron graft was 9.63 ± 0.53 CFU/mL. Antibiotics achieved a mean reduction of 48%, with ciprofloxacin performing best (67% reduction). Baseline MSSA6 count on the Dacron-silver-triclosan graft was 8.54 ± 0.73 CFU/mL. Antibiotics reduced biofilm formation by 72%; cloxacillin was the most effective treatment (86%). The MSSA6 count on the untreated Omniflow-II graft was 1.17 ± 1.52 CFU/mL. For the bovine pericardium patch, it was 8.98 ± 0.67 CFU/mL. Mean reduction after antibiotic treatment was 46%, with cloxacillin achieving the greatest reduction (68%). In this in vitro study, ciprofloxacin and cloxacillin performed best at reducing biofilms formed by clinical MSSA strains on the surface of biological and synthetic vascular grafts.