Given the contradictory nature of the literature regarding the role of human papillomaviruses and polyomaviruses in the pathogenesis of urothelial bladder cancer (UBC), we sought to investigate the frequency of their involvement in a large cohort of primary UBCs. DNA was extracted from 689 fresh-frozen UBC tissues and screened for the presence of high-risk human papillomavirus (HPV) types 16 and 18 and BKV/JCV genomic DNA by qPCR. In positive cases, viral identity was confirmed by Sanger sequencing and viral gene expression was analysed by RT-PCR or immunohistochemistry. All 689 UBCs were negative for HPV18. One UBC from a female patient with areas of squamous differentiation was positive for HPV16. The qPCR data indicated variable levels of polyomavirus in 49 UBCs. In the UBCs with low Cts we were able to confirm that 23 were BKV and 6 were JCV by Sanger sequencing. Polyomavirus large T antigen expression was low but detectable in 70% of the sequencing-confirmed polyomavirus positive samples. Thus, in United Kingdom patients, the presence of HPV DNA sequences is extremely rare in UBC (<1% of cases). Polyomavirus DNA (predominantly BKV) is more common in UBC, but still only detectable in 7% of cases and in many of these cases at low copy number. We have performed the largest virus screening to date in UBC, finding that HPV16, HPV18 and HPyV are unlikely to be common causative agents in UBC.