An infectious etiology for bladder cancer has long been suspected. Merkel cell virus (MCV), BKV and JCV polyomaviruses are possible causative agents but data remain scarce. Therefore, we evaluated the seroresponse to these three polyomaviruses in association with bladder cancer risk. 1,135 incident bladder cancer subjects from five Spanish regions and 982 hospital controls matched by sex, age and region were included. 99% of cases were urothelial-cell carcinomas. Antibody response against MCV, BKV and JCV was measured by enzyme immunoassay using Virus-Like-Particles. Our results show a similar seroprevalence in cases and controls: 64/60% for BKV, 83/82% for MCV and 87/83% for JCV. However, among seropositive subjects, higher median seroreactivities were observed in cases compared to controls for BKV (0.84 vs. 0.70, p-value = 0.009) and MCV (1.81 vs. 0.65, p-value < 0.001). Increased bladder cancer risk was observed for BKV (OR = 1.4, 95%CI 1.04–1.8) and for MCV (OR = 1.5, 95%CI 1.2–1.9), when comparing highest to lowest seroreactivity tertiles. The associations of BKV and MCV with bladder cancer were independent of each other and neither smoking status nor disease stage and grade modified them. Furthermore, no association was observed between seroresponse to JCV and bladder cancer. Therefore, we conclude that BKV and MCV polyomavirus infection could be related to an increased bladder cancer risk.