Mastitis is one of the most important diseases in dairy cattle of which Staphylococcus aureus is a major pathogen. Despite an apparently good antimicrobial susceptibility in vitro, the cure of diseased animals from this bacteriological infection is often disappointing, which results in cases of recurrent clinical- and chronic subclinical infections. It has been suggested that these recurrent and chronic Staphylococcus infections can be attributed to the growth of bacteria in biofilm. The objective of this study was to compare the susceptibility for antimicrobial agents of S. aureus isolates obtained from bovine mastitis growing under different conditions. These conditions include a conventional conventional microbroth dilution assay in which minimal inhibitory concentration values are determined, the MBEC assay which measures both the susceptibility in biofilm and the susceptibility of sequester cells released from the biofilm. A comparison of the susceptibility for antimicrobial agents of a number of representative S. aureus isolates grown in broth (representing in vitro growth conditions) or milk (representing in vivo growth conditions) is also made. The results indicate that S. aureus isolates obtained from bovine mastitis are highly resistant to antimicrobial agents when growing in biofilms.