Rapid and precise detection of pathogenic microbes particularly gram-positive pathogens possibly Staphylococcus aureus is essential in the fields of microbiology and food security. Public safety problems are raised by outbreaks of foodborne diseases, which are frequently caused by sources originating from animals and involve pathogens such as Staphylococcus aureus and Listeria monocytogenes. To stop outbreaks, it is essential to closely monitor raw food for the presence of these infections. Gram-positive Staphylococcus aureus is a serious danger because it produces heat-stable staphylococcal enterotoxins (SEs), which can lead to serious foodborne diseases. These toxins serve as strong gastroenterotoxins and superantigens, which emphasizes the necessity of effective detection techniques. This chapter introduces multiplex polymerase chain reaction (multiplex PCR) as an advanced and efficient method for the precise identification of S. aureus and its associated enterotoxins. Multiplex PCR streamlines detection by amplifying multiple target genes simultaneously, addressing the adaptability of S. aureus to diverse food processing conditions.