Abstract Background: Salmonella spp. and Escherichia coli O157:H7 are the most common bacterial foodborne pathogens contaminating food products especially meat. It is essential to detect the pathogens rapidly, specifically, and simultaneously by selection and optimization of suitable reference genes. The present study was conducted to simultaneously detect E. coli O157:H7 and Salmonella spp. in meat products and contamination prevalence assay by using multiplex PCR based on rfbE and invA genes amplification in Zanjan province, northwest of Iran. Materials and Methods: A total of 74 meat samples were obtained from various regions of Zanjan province, randomly. 25 grams of each meat sample was completely homogenized in 225 ml of Mueller-Hinton broth growth medium and incubated. Bacterial strains were purified and DNA extraction was performed from purified bacterial isolates. Simultaneous amplification of rfbE and invA gene fragments was done with specific primers by optimization of a multiplex PCR. Finally, the sensitivity of the method was evaluated by inoculation of the bacteria to the meat. Results: Out of 74 meat samples, 6(8%), 4(5.4%), and 2(2.7%) samples were positive for E. coli O157:H7, Salmonella, and both E. coli O157:H7-Salmonella, respectively. Multiplex PCR indicated high sensitivity for simultaneous detection of the pathogens in lowest dilution of the bacteria that had been inoculated to the meat. Conclusion: In this study, a multiplex PCR was optimized based on Salmonella spp. and E. coli O157:H7 virulence genes for rapid and simultaneous detection of the pathogens with high sensitivity and specificity. Multiplex PCR as a reliable tool for rapid and simultaneous detection of foodborne pathogens to prevent contamination of food products.