Although several cases and case-control studies reported the prevalence of JC polyomavirus (JCV) and BK polyomavirus (BKV) in tumor tissues of colorectal cancer (CRC) patients as well as in control samples; however, many recent studies did not show the prevalence of these viruses in the CRC patients. A total of 50 clinical biopsies samples were collected from patients with and without CRC in the General surgery department, Faculty of Medicine, Ain Shams University, Egypt, with the approval of the research ethics committee. Current results showed the absence of the genomic DNA of both JCV and BKV in the tumor tissue samples, adjacent-tumor normal mucosa samples from CRC patients, and also in the normal mucosa samples collected from the non-CRC patients. Moreover, results of the semi-nested PCR and real-time PCR are similar, and confirmed that JCV and BKV are not prevalent in the Egyptian CRC patients. There are no promising results for further investigation of the expression of the large T-Ag protein, and any other related proteins in the tumor cells. Based on the present results, no evidence supports the association between the JCV, BKV and CRC in the Egyptian patients. For the best of our knowledge, this is the first case-control study on the prevalence of JCV and BKV in the Egyptian CRC patients. The aim of the current study was to detect the presence of JCV and BKV genomic DNA in the tissue samples, to support further studies on the role of these viruses in the CRC etiology and development.