In the present study, we developed a gold nanoprobe assay, which relies on the colorimetric differentiation of specific DNA sequences, based on different aggregation profiles. We evaluated the assay on DNA extracted from pathogen cultures and from contaminated food specimens. The targets used were the femA gene for the identification of Staphylococcus aureus, the hly gene of the Listeria monocytogenes listeriolysin and the invA sequence for Salmonella spp. Comparison was performed with the reference assay, as described in the respective ISO guidelines for each pathogen, and a direct PCR amplification and detection. The minimum detection limit of the assay was defined at 123 f g/ μ L DNA, for all species, lower than PCR detection. Specificity was 100%. Sensitivity was 100%, as compared το the reference method, whereas the sensitivity of PCR was 93.3%. The evaluated assay could be used as a sensitive screening method, which does not require major infrastructure, for the detection and identification of pathogens in food specimens. In addition, it can accommodate detection of multiple species, thus allowing multiplexing and expedite turnaround time.