ABSTRACTYersinia enterocolitica, Staphylococcus aureus, Aeromonas and Salmonella are among the most important foodborne bacterial pathogens. The majority of human infections caused by all of these organisms are associated with ingestion of undercooked and contaminated meat, dairy products and water where in the secreted bacterial toxins lead to foodborne intoxications. We, here, report a new multiplex polymerase chain reaction (mPCR) assay for the simultaneous detection of these important foodborne bacterial pathogens. The mPCR targeted Ail and virF genes of Y. enterocolitica, nuc and entB genes of S. aureus, aerA and 16S rRNA genes of Aeromonas and invA, an invasion protein A gene of Salmonella. An internal amplification control designed to check the false negative reactions in mPCR was also included. This procedure could detect initial populations of 1–100 cfu/g or /mL within 24 h in experimentally spiked food and water samples. When evaluated on a total of 104 naturally occurring food samples, the mPCR detected two samples to contain S. aureus, one was identified to contain Y. enterocolitica and four samples were identified to contain Salmonella species individually. This was compared with the standard microbiological and biochemical identification procedures.PRACTICAL APPLICATIONSAll the microorganisms selected in this study are food and waterborne and contaminate a variety of food items. Pathogenic Y. enterocolitica and Aeromonas species are able to grow and multiply and secrete toxins even at low temperatures. The high throughput and cost‐effective multiplex polymerase chain reaction method reported here could be a viable alternative for detection of pathogenic Y. enterocolitica, S. aureus, Aeromonas and Salmonella from food and environmental samples.