Objectives: Human papillomavirus (HPV) is a well-known oncogenic virus associated with anogenital carcinomas. Despite the anatomical proximity of the bladder and the anogenital region, the relationship between HPV and urothelial carcinoma of the bladder (UCB) is still a controversial issue. This study aimed to test the urethral swabs and first-void urine samples of patients with UCB for HPV-Deoxyribonucleic acid (DNA) using polymerase chain reaction (PCR) assay and to compare the results with a control group. Materials and Methods: Sixty-nine patients who were diagnosed with UCB between January and December 2018 were included in this case-control study. Sixty-nine patients who visited the urology outpatient clinic for non-oncological reasons within the study period were designated as the control group. Urethral swab and first-void morning urine samples were collected from each patient. HPV-DNA presence was investigated using a PCR kit that can detect a total of 22 HPV genotypes, of which 18 are high-risk and 3 are low-risk genotypes. Results: The mean age of the patients included in the study was 63.2 +/- 12.6 years and the male to female ratio was 5.3. HPV-DNA was detected in 28.9% (20/69) of the patients in the case group and in 8.7% (6/69) of the patients in the control group. HPV-DNA positivity was significantly higher in the case group (OR 4.24; 95% CI 1.63-12.34). No statistically significant relationship was found between HPV-DNA positivity and tumor grade (p = 0.36). Conclusion: A statistically significant relationship exists between HPV infection and UCB, regardless of the tumor grade.