ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding alpha-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently, expression of all three genes were repressed by aminoglycosides and induced by fluoroquinolones and penicillins. In contrast, the beta-lactam sub-group cephalosporins enhanced expression of RNAIII and hla but diversely affected expression of spa. The compounds cefalotin, cefamandole, cefoxitin, ceftazidime and cefixine were found to up-regulate spa, while down-regulation was observed for cefuroxime, cefotaxime and cefepime. Interestingly, biofilm assays demonstrated that the spa-inducing cefalotin resulted in less biofilm formation compared to the spa-repressing cefotaxime. CONCLUSIONS: We find that independently of the cephalosporin generation, cephalosporins oppositely regulate spa expression and biofilm formation. Repression of spa expression correlates with the presence of a distinct methyloxime group while induction correlates with an acidic substituted oxime group. As cephalosporines target the cell wall penicillin binding proteins we speculate that subtle differences in this interaction fine-tunes spa expression independently of agr.