Biofilm formation by Staphylococcus aureus is a serious problem in nosocomial infections. There are great differences in the capacity of S. aureus to express biofilms, but the reasons are unknown. In all, 105 S. aureus strains were tested for a correlation between the agr quorum-sensing system phenotype and the ability of S. aureus to adhere to polystyrene. Some 78% of agr-negative, but only 6% of agr-positive, strains formed a biofilm, demonstrating a profound impact of agr on biofilm formation. This result was confirmed with defined agr mutants and by inhibition of agr with quorum-sensing blockers. The observed effect was not due to differential expression of the autolysin Atl or of the exopolysaccharide polysaccharide intercellular adhesin but seemed to be caused, at least in part, by the surfactant properties of delta-toxin. The detected biofilm-enhancing effect of S. aureus quorum-sensing blockers call into question the proposed therapeutic use of such substances.