Staphylococcal enterotoxin D and R (SED, SER) production was determined in 24 S. aureus strains harboring sed gene. Seven of them were not able to produce SED as evidenced by enzyme-linked immunosorbent assay and Western blotting. Sequencing revealed that all these strains harbor a variant of sed gene. Expression of SER was detectable in 22 out of 24 isolates, with variance in productivity ranging from ∼40 to 450 ng/mL. Out of the seven isolates not able to produce SED, three produced high amounts of SER (249-396 ng/mL), two produced less than 200 ng/mL of SER, and two were found to express no detectable amount of SER. Three of those were assigned to spa type t1677 with two being of agr type III and one of agr type I. One strain was t084, agr type II, one t603, agr type II, one 2920, agr type III, one t2920, agr type III, and one t5160, agr type I. Because conventional screening procedures involve only the detection of classical enterotoxins in food, the isolates not able to produce SED presented in this study could pose a threat to human health due to SER production.