Polyomaviruses such as the BK virus (BKV), JC virus (JCV) and SV40, as well as the human papillomaviruses (HPV) are frequently detected throughout human populations, causing subclinical persistent infections and inducing oncogenesis in human and other cell lines. To test the involvement of these viruses in prostate tumorigenesis, we investigated the prevalence of BKV, JCV and HPV in a series of human prostatic malignancies. Forty-two samples of diagnosed prostatic malignancies were tested using standard polymerase chain reaction (PCR) protocols. Differentiation between BKV and JCV among the polyomavirus-positive samples was achieved after sequencing analysis of the PCR products. Reconstitution of BKV in vitro was performed and indirect immunofluorescence for the large T-antigen of the virus was applied to confirm the production of progeny virus. Detection and typing of HPV was carried out by PCR. The overall prevalence of polyomaviruses was 19% in the prostate cancer cases. Sequencing analysis of the polyomavirus-positive specimens revealed the presence of BKV in all samples. Reconstitution of the BKV from the BKV-positive prostate samples was successfully achieved in cell culture and progeny viral particles were obtained, confirming the presence of the virus in the human biopsies. HPV was detected in 4.8% of the samples, however, no HPV-11, HPV-16, HPV-18 or HPV-33 types were identified. BKV was frequently detected and could play a relevant role in the development and progression of human prostate cancer, whereas HPV does not seem to be implicated in this type of human neoplasia.