We studied 358 Staphylococcus aureus strains isolated from bloodstream infections (BSI) observed during an epidemiological study covering 2,007,681 days of hospitalization in 32 healthcare institutions (HCIs) between 2004 and 2006. The strains were tested for antibiotic susceptibility and characterized genetically. The incidence of S. aureus BSI declined regularly through 2004 and 2005 and then significantly increased in 2006 (+80%). This was largely due to an increase in BSI involving methicillin-sensitive S. aureus (MSSA) strains and nonmultiresistant methicillin-resistant S. aureus (NORSA) strains. Ninety-six percent of the NORSA strains were resistant only to methicillin and fluoroquinolones. Most of the MSSA strains belonged to a small number of pulsed-field gel electrophoresis (PFGE) divisions and were associated with epidemic phenomena in HCIs. The NORSA strains also clustered into a limited number of PFGE divisions but could not be related to any local outbreak in HCIs. In 2006, there was a significant increase in the incidence of BSI associated with tst gene-positive MSSA strains (+275%) and the first three BSI associated with tst gene-positive MRSA were observed. PFGE data revealed a limited heterogeneity among the tst gene-positive strains without any outbreak in the HCIs. Our study underlines the need for infection control teams to focus efforts on preventing both MRSA and MSSA BSI. As recently demonstrated in vitro, fluoroquinolones may enhance horizontal transfer of virulence and antibiotic resistance genes. These antibiotics are widely used in France, so our findings raise the issue of whether their use has contributed to the acquisition of mecA and tst genes by S. aureus strains.