The relationship between urothelial bladder cancer and high-risk human papillomaviruses (HR-HPV) is still a poorly understood entity, even if some studies have supposed a probably correlation. The aim of the present study was to assess the potential relationship between the presence of HR-HPV and non-muscle invasive urothelial bladder cancers (NMIBC). One hundred and thirty-seven subjects (78 patients affected by NMIBC and 59 controls) were recruited in this prospective study. HR-HPV DNA was evaluated both in urine and tumour tissues. Data from patients were compared with data from controls. The relationship between patients and controls, in terms of HR-HPV presence was performed. The relationship between all pathological data and HR-HPV presence in patient group was carried out. HR-HPV DNA in tissue was found in 27 of 78 (34.6%) tumour samples and in 6 of 59 (10.1%) specimens from TUR-P, with a statistically significant difference (p=0.0009; dF=1; χ2=10.98). HR-HPV DNA in urine was found in 36 of 78 (46.1%) samples obtained from patients, whereas in only 8 of 59 (13.5%) samples from controls (p<0.0001: dF=1; χ2=16.37). A statistical significant difference in terms of HR-HPV frequency between high-grade and low-grade urothelial bladder cancer, was found (p=0.032; RR=0.52-95% CI 0.27-0.93; OR=0.34-95% CI 0.13-0.90). In conclusion, this study highlights the correlation between urothelial bladder cancer and high-risk type HPV infection, suggesting the potential etiopathogenetic role of HR-HPV in urothelial bladder cancer development.