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This study evaluated vancomycin susceptibility and activity alone and in combination with rifampicin and tigecycline against low-biofilm- and high-biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates.Forty MRSA isolates recovered from bloodstream infections were analysed. Susceptibilities were performed in planktonic and biofilm cultures by microbroth dilution. Biofilm production was determined using an adherent plate assay. Time–kill analysis was performed on six low- and six high-biofilm-producing isolates with 15 mg/L vancomycin alone and in combination with rifampicin or tigecycline at 4× MIC.Vancomycin susceptibility displayed a 4-fold and an 8-fold increase in the MIC50 and MIC90, respectively, in the presence of biofilm. Rifampicin and tigecycline susceptibilities also increased in biofilms, but still remained within the susceptibility breakpoints except for a tigecycline MIC90 of 1 mg/L. High biofilm production was detected in 60% of the isolates. In time–kill analysis, 15 mg/L vancomycin achieved bactericidal activity against only low-biofilm-producing strains with a 1.8 log10 cfu/mL difference in bacterial kill compared with high-biofilm-producing strains (P < 0.001). Rifampicin alone had minimal activity, resulting in resistance. Tigecycline was minimally effective and was not bactericidal, but no difference was observed in the comparison of biofilm-producing strains. Vancomycin in combination with rifampicin or tigecycline was bactericidal against all strains (mean kill 4.5 ± 0.5 log10 cfu/mL), regardless of biofilm production.Vancomycin exposures at 15 mg/L may not be adequate in eradicating biofilm-producing S. aureus. Alternative treatments or combination therapy should be explored to optimize outcomes in biofilm-associated infections.