The aim of this study was to determines the prevalence of virulence gene hemolysin A (hly A) Escherichia coli and Staphylococcal enterotoxins (sea) in Staphylococcus aureus in raw milk buffaloes. In molecular laboratory, real-time polymerase chain reaction (RT-PCR) technique has been performed for 24 samples which have been taken randomly from Buffaloes milk, using primers of high specificity for Escherichia coli hlyA gene and Staphylococcus aureus Sea genes. The results showed different degrees of the studied genes activities. Four out of 24 samples represented S. aureus Sea gene (16.6%) whereas 16 out of 24 samples represented E. coli hlyA gene (66.6%). this study concluded that buffaloes milk might be a source of contamination with pathogenic bacteria of virulent genes which may have different levels of activities.