Background and Objectives: Salmonella typhi, Bacillus anthracis, and Yersinia pestis are potent human pathogenic bacteria that cause typhoid fever, anthrax, and plague,respectively. The classic microbiological methods for detection and identification of these agents are laborious and time consuming.Therefore, developing an accurate method for rapid detection of these potent human pathogens is important, especially due to their potential use as Bioterrorism agents. We have designed a new rapid molecular detection method by using multiplex PCR in a single reaction. Materials and Methods: PCR was carried out using specific primers for the virulence factor of S. typhi, hp and invA, B. anthracis virulence factors, chr and pa, and Y. pestis toxin, pla. The primer sets were tested for cross reactivity with individual DNA strains and mixtures in a multiplex PCR format. For determination of sensitivity of the method, we used colony counting and genomic DNA dilution. Results: Theresult with standard strains revealed that primers are specific for each strain as they have successfully amplified the desire products. The sensitivity analysis showed a 1-10 CFU and 1-10 copies of the genomes. In this respect, the accuracy of the result was checked by sequencing of the 1083-bp PCR products, except for anthrax, which was determined by enzymatic digestion. Conclusion: We have found a rapid, specific, sensitive, and a non-expensive molecular method for detection of three potent human pathogens. This finding provide an efficient diagnostic tool to determine these bacteria in bioterrorism samples.