The MBCs of three commonly used hospital biocides [containing quaternary ammonium compounds (QACs), chlorhexidine gluconate and triclosan] were determined for clinical isolates of Staphylococcus aureus, which were also screened for genes encoding Qac efflux pumps. MBCs were determined by broth microdilution for 94 clinical isolates of S. aureus, including 38 hospital-acquired methicillin-resistant S. aureus (HA-MRSA), 25 community-associated methicillin-resistant S. aureus (CA-MRSA), 25 methicillin-susceptible S. aureus (MSSA) and 6 with intermediate resistance to vancomycin (VISA). All isolates were screened by PCR for the presence of qacA, B, C, G, H and J. Biocides had MBCs 10-1000-fold lower than the concentration recommended for use by the manufacturer. HA-MRSA isolates developed significantly enhanced tolerance to QACs following repeat exposure to subinhibitory concentrations. Ten HA-MRSA and four VISA isolates carried qacA. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried qacC. One VISA isolate carried qacA and qacC. The CA-MRSA isolates did not carry qac genes. qacG, H and J were not detected in any HA-MRSA. Isolates with qac genes had significantly (P < 0.0001) higher MBCs for biocides containing QACs and chlorhexidine gluconate. These biocides induced expression of qac genes when assayed with a luciferase reporter. Biocides commonly used in the hospital environment should be effective against clinical isolates of S. aureus if used at concentrations recommended by the manufacturer. However, isolates have the potential to develop increased tolerance to these agents and the expression of Qac efflux pumps results in isolates with a selective advantage when challenged with biocides containing QACs and chlorhexidine gluconate.