Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects were analyzed for immunoglobulin (Ig) G binding to 20 toxins, using flow-cytometry based technology. Antibody levels were associated with polymerase chain reaction-defined presence of toxin genes in homologous S. aureus isolates. IgG levels directed to exfoliative toxin (ET) A, ETB, ghemolysin B (HlgB), leukocidin (Luk) D, LukE, LukS, staphylococcal enterotoxin (SE) A, SEE, SEH, SEI, and SElM were higher in S. aureus-infected patients than in control subjects (P <.05). Furthermore, in the S. aureus-infected patient group, IgG levels were higher if genes encoding ETA, ETB, SEA, SEC, SEH, SElQ, toxic shock syndrome toxin-1 (TSST-1), or Panton-Valentine leukocidin (PVL) were present in the infectious isolate (P < .05). Levels of anti-SEA IgG increased during infections with sea-positive (median fluorescence intensity from 11,555 to 12,388; P<.05) but not sea-negative strains. In addition, anti-LukS IgG levels increased during skin and soft-tissue infections with luk-PV-positive (median fluorescence intensity from 15,231 to 15,911; P < .05) but not luk-PV-negative strains. Bacteremia was associated with sea (odds ratio, 3.4; 95% confidence interval, 1.2–10.0) and tst (odds ratio, 5.7; 95% confidence interval, 1.6–20.8). Skin and soft-tissue infections and bone and joint infections were associated with luk-PV (odds ratio, 2.5; 95% confidence interval, 1.2–5.2). Many toxins are expressed in vivo and recognized by the immune system during staphylococcal infections, suggesting their involvement in S. aureus pathogenesis.