A total of 100 minced meat samples were collected from different butchers in Port-Said city for isolation and identification of Staph. aureus, Escherichia coli and Salmonella spp.. The results revealed that Staph. aureus, E. coli and Salmonella spp. could be detected in a percentage of 14%, 12% and 1% respectively from minced meat samples. Molecular characterizations of all isolated S. aureus, E. coli and Salmonella spp. were confirmed using 16S rRNA, phoA and invA, respectively by conventional PCR at 791 bp; 720 bp and 284 bp, respectively. Multiplex PCR was developed with specific primers for the detection of different enterotoxin genes (Sea, Seb, Sec, Sed and See) of Staph. aureus, (Stx1, Stx2, STa and lt) of E. coli and (stn) of Salmonella which may be considered a significant in food safety threat. The obtained results showed that the positive serotypes for enterotoxin genes were (Seb in 3 isolates and Sed in one isolate) of Staph. aureus at 164 bp and 278 bp respectively; (Stx2 in 2 isolates and STa in 2 isolates) of E. coli at 779 bp and 229 bp, respectively. Conventional PCR is rapid methods for the confirmation of Staph. aureus, E. coli and Salmonella spp., while multiplex PCR is a useful technique for detection of enterotoxin genes. The public health hazards of this isolated organism, as well as recommended measures to improve quality status of minced meat were discussed.