A total of 429 different Staphylococcus aureus isolates encompassing 219 blood isolates and 210 isolates taken from anterior nares were systematically searched by two multiplex PCR-DNA enzyme immunoassays (PCR-DEIA) for exfoliative toxin (ET) genes eta and etb, as well as for the classical members of the pyrogenic toxin superantigen (PTSAg) gene family comprising the staphylococcal enterotoxin (SE) genes sea-see and the toxic shock syndrome toxin 1 gene tst. In addition, a third PCR-DEIA was established to investigate the possession of four recently described SE genes, viz. seg-sej. The most frequent PTSAg/ET genes amplified were seg and sei, which were found strictly in combination in 55.0% of the S. aureus isolates tested. Other frequently detected toxin genes were tst (20.3%), sea (15.9%), and sec (11.2%). Only five isolates harbored ET genes. Regarding the origin of the S. aureus isolates, a significant difference (P = 0.037) was found for the possession of the sed/sej gene combination (10.5% of blood isolates versus 3.3% of nasal strains). Overall, about half of S. aureus isolates tested harbored genes of the classical members of the PTSAg family and ETs (50.8%), whereas 73.0% of S. aureus isolates were toxin gene positive if the recently described SE genes were included. This notable higher prevalence indicates that the possession of PTSAg genes in particular seems to be a habitual feature of S. aureus. Moreover, mainly due to the fixed combinations of seg plus sei, as well as sed plus sej, the possession of multiple PTSAg genes (62.9%) is more frequent than assumed so far.