To investigate the role of a putative small multidrug resistance transporter, annotated in Enterococcus faecalis V583 genome as EFA0010 (we will refer to this gene as qacZ), in decreased susceptibility to biocides. A derivative strain of V583, susceptible to erythromycin (V583ErmS), was complemented with pORI23 carrying the qacZ gene (strain EF-SAVE1). MICs of benzalkonium chloride, chlorhexidine and ethidium bromide were determined for the complemented strain and wild-type. RT-PCR and ethidium bromide efflux assays were performed in order to fully understand the role and specificity of the qacZ gene. The presence of qacZ in 73 enterococcal strains from different origins was investigated by PCR, and MICs of benzalkonium chloride and chlorhexidine were determined for the same strains. The complemented strain, EF-SAVE1, presented a higher MIC of benzalkonium chloride (8 mg/L) than V583ErmS (4 mg/L); the MICs of chlorhexidine and ethidium bromide were the same for both strains, 4 mg/L and 16 mg/L, respectively. Expression of qacZ was found to be higher in EF-SAVE1 and constitutive, i.e. not inducible by any of the three tested biocides. Overexpression of qacZ was not responsible for changes in ethidium bromide efflux. This gene was present in 52% of the enterococcal isolates studied and the MICs of benzalkonium chloride and chlorhexidine ranged between 2 and 8 mg/L. We demonstrate the involvement of the qacZ gene in tolerance to the quaternary ammonium compound benzalkonium chloride, but not ethidium bromide. This work constitutes the first report of a biocide resistance mechanism in E. faecalis, and reveals its dissemination amongst the genus Enterococcus.