Staphylococcal Enterotoxin B (SEB) and Shiga Toxins (STX1 and STX2) pose significant threats to food and water safety and are associated with bacterial strains that are considered to be potential bioterrorism agents (BA). Given their public health relevance and misuse potential, the rapid and accurate detection of toxin genes is essential for effective surveillance and response. The Brazilian Army plays a crucial role in biodefense efforts, including BA identification methods. This study focused on developing a multiplex qPCR assay for seb, stx1, stx2, and 16S rRNA gene targets. The design of the Stx2 primer was performed using PrimerQuest Tool; its specificity was evaluated in Primer-BLAST, and its secondary structure was analyzed using the OligoAnalyzer Tool. Staphylococcus aureus ATCC 25923, S. aureus NCTC 12493, Escherichia coli O157:H7, and E. coli ATCC 25922 strains were used for validation tests. Bacteria were grown in BHI media and incubated at 37 °C for 24 h. DNA extraction was performed using the MPTA016 pathogen kit in the Extracta32 system, and amplification was carried out on the Amplio96™ equipment. The probe concentration and DNA mass were 250 nM and 10 ng, respectively. The optimal temperatures revealed were 58.4 °C, 60.2 °C, and 58.4 °C for stx1 (Cq=22.49), seb (Cq=26.10), and 16S rRNA (Cq=14.91), respectively, supporting their inclusion in a multiplex setup. Therefore, 58.4 °C was selected for the assay. The 16S rRNA primer concentration showed no significant difference between 250 nM and 350 nM, allowing for the selection of 250 nM. For stx1, 350 nM provided a superior performance. No amplification was observed in non-toxin-producing strains, confirming the stx1 and seb primers' specificity. The stx2 primer and probe exhibited melting temperatures of 62 °C and 67 °C, respectively. Only heterodimer formation was observed (–13.29 kcal/mol). Our results showed that the primers were specific, allowing for their application to identifying the target genes proposed. This protocol was integrated into the Brazilian Army's biodefense system workflows to enhance its national and military biosurveillance.