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Brucella spp. and Listeria monocytogenes are recognised as important food safety and public health concerns. We provide a unique single-tube real-time platform for simultaneous yet differential detection of Brucella spp. and L. monocytogenes in milk using melt curve analysis. Melting curve analysis revealed two different peaks at melting temperatures of 79.91 ± 0.56 °C and 87.51 ± 0.26 °C for L. monocytogenes and Brucella spp., respectively. The technique detected only L. monocytogenes and Brucella spp., with no different melt curves for other bacterial pathogens. The limit of detection (LoD) in spiked raw milk demonstrated that the duplex test detects the target pathogens up to 10 cfu mL−1 milk. The findings show that the newly proposed method is easy-to-use, sensitive, specific, and a viable diagnostic tool for testing a wide range of foods that are concomitantly contaminated with L. monocytogenes and Brucella spp. [ABSTRACT FROM AUTHOR]