Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.