Raw cow milk is considered one of the most important vehicles for pathogenic bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. These three bacteria are responsible for foodborne diseases. Routinemicrobiologicalmethodstodetectthesemicroorganismsin cowmilkcanbecomplicatedandtimeconsuming. Theaimofthisworkwas toevaluateamethodtosimultaneouslydetectSalmonellaspp.,Escherichiacoli O157:H7, and Listeria monocytogenes in experimentally contaminated cow milk. The assessed method combined a standard microbiological culture step, using a pre-enrichment medium that favors the growth of the three focal microorganisms: SEL broth, followed by a single PCR assay. A total of 43 interference bacterial strains were used to evaluate the method’s specificity. The detection rate for the microbiological method with standard culture media was 10 UFC/mL, and that of the PCR detection, following pre-enrichment in SEL broth, was 10 UFC/mL for S. enterica and L. monocytogenes and between 1 and 5 UFC/mL for E. coli O157:H7. The PCR method showed specificity for the reference strains. Simultaneous detection by multiple PCR using SEL broth was successful for thedetectionofS.enterica,E.coliO157:H7,andL.monocytogenesinsamples of experimentally contaminated cow milk, featuring both a high detection rate and a high specificity. This approach promises to be a feasible routine procedure when testing milk samples in industry and public health control setups.