Abstract Background Seroepidemiological studies have reported associations between exposure to sexually transmitted organisms and prostate cancer risk. This study sought DNA evidence of candidate organisms in archival prostate cancer tissues with the aim of assessing if a subset of these cancers show any association with common genital infections. Methods 221 archival paraffin-embedded tissue blocks representing 128 histopathologically confirmed prostate cancers comprising 52 “aggressive” (Gleason score ≥ 7) and 76 “non-aggressive” (Gleason score ≤ 6) TURP or radical prostatectomy specimens were examined, as well as unaffected adjacent tissue when available. Representative tissue sections were subjected to DNA extraction, quality tested and screened by PCR for HSV-1, HSV-2, XMRV, BKV, HPV, Chlamydia trachomatis, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Trichomonas vaginalis . Results 195 of 221 DNA samples representing 49 “aggressive” and 66 “non-aggressive” prostate cancer cases were suitable for analysis after DNA quality assessment. Overall, 12.2% (6/49) aggressive and 7.6% (5/66) non-aggressive cases were positive for any of the candidate organisms. Mycoplasma genitalium DNA was detected in 4/66 non-aggressive, 5/49 aggressive cancers and in one cancer-unaffected adjacent tissue block of an aggressive case. Ureaplasma urealyticum DNA was detected in 0/66 non-aggressive and 1/49 aggressive cancers and HSV DNA in 1/66 non-aggressive and 0/49 aggressive cancers. This study did not detect BKV, XMRV, T. vaginalis , U. parvum , C. trachomatis or HPV DNA. Conclusions The low prevalence of detectable microbial DNA makes it unlikely that persistent infection by the selected candidate microorganisms contribute to prostate cancer risk, regardless of tumour phenotype.