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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, <i>sea, seb</i>, <i>sec, sed</i>, and <i>see</i>, among <i>Staphylococcus aureus</i> 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 <i>S. aureus</i> (<i>n</i> = 51) and CNS (<i>n</i> = 47). The expression of classical enterotoxin genes, including <i>sea, seb, sec,</i> and <i>see</i>, 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 <i>S. aureus</i> (35.30%) than in CNS (12.77%). The <i>sec</i> gene was most frequently detected in <i>S. aureus</i> (29.41%) and CNS (6.38%). Moreover, the expression of <i>sea</i> and <i>sec</i> was significantly higher at room temperature than at 8 °C after 16 h of incubation (<i>p</i> < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.