We developed a multiplex PCR (mPCR) assay to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Corynebacterium spp. and seudomona aeruginosa. This method employs a single tube and multiple specific primers which yield 200, 281, 346, 423, 542, and 1,427 bp PCR products, respectively. All the PCR products were easily detected by agarose gel electrophoresis and were sequenced to confirm the specificity of the reactions. To test this method, DNA extracted from urine samples was collected from 96 sexually transmitted disease or prostatitis patients at a local hospital clinical center, and were subjected to the mPCR assay. The resulting amplicons were cloned and sequenced to exactly match the sequences of known pathogenic isolates. N. gonorrhoeae and Corynebacterium spp. were the most frequently observed pathogens found in the STDs and prostatitis patients, respectively. Unexpectedly, P. aeruginosa was also detected in some of the STD and prostatitis samples. More than one pathogen species was found in 10% and 80.7% of STD and prostatitis samples, respectively, indicating that STD and prostatitis patients may have other undiagnosed and associates. The sensitivity of the assay was determined by sing purified DNA from six pathogenic laboratory strains and revealed that this technique could detect pathogenic DNA at concentrations ranging from 0.018 to $1.899;pg/{mu}l$. Moreover, the specificities of this assay were found to be highly efficient. Thus, this mPCR assay may be useful for the rapid diagnosis of causative infectious STDs and prostatitis. useful for the infectious STDs and prostatitis.