Strains of diarrheagenic Escherichia coli (DEC) are involved in foodborne disease outbreaks worldwide, especially the enterohemorrhagic E. coli O157:H7. This study describes two multiplex quantitative real time PCR (qPCR) assays for simultaneous identification and quantification of genes related to virulence of DEC; a triplex reaction for detection and quantification of stxA1, stxA2, and eaeA genes, and a duplex reaction for detection and quantification of eaeA and virA genes. The technique was applied in raw oyster samples for direct quantification of DEC, thereby evaluating the applicability of this methodology for microbiological quality assessment of food. Using custom designed primers and specific MGB probes, a triplex qPCR assay was performed to quantify stxA1, stxA2, and eaeA, and a duplex reaction was performed to quantify virA and eaeA genes. The assays showed high sensitivity, with the detection limit varying between 5 and 17 copies of the genes. The coefficient of determination (R2) of the standard curves was 0.99. The coefficient of variation was < 1% indicated high intra- and inter-assay reproducibilities. The application of this methodology in oyster samples from tropical environment provided direct quantitative data that determined the presence of the genes stxA1 (32.1%), eaeA (28.6%), stxA2 (3.6%), and virA (3.6%). This would prove critical for immediate intervention of control strategies, particularly in oysters that are often ingested as raw food.