Infertility, affecting approximately 16% of the global population, has led to increased reliance on reproductive medicine. The impact of human papillomavirus (HPV) infection in one or both partners on outcomes of Assisted Reproduction Technologies (ART) remains unclear. This prospective cohort study aimed to evaluate prevalence and effects of HPV infection in subjects and couples candidates to ART. A total of n = 510 men and n = 246 women were included and n = 145 couples (n = 290 individuals) had both partners enrolled in the study. The HPV semen infection rate was 17% (95% CI: 14–20) with HPV‐42, HPV‐16, HPV‐53 and HPV‐51 as the most frequently detected genotypes. In women, 26% (95% CI: 21–32) tested HPV‐positive in cervical swabs. In 6% (95% CI: 3–11) of the couples, both partners were positive but only three couples shared the same genotypes (HPV‐16; HPV‐39, HPV‐51, and HPV‐42; HPV‐31). Follicular fluids were positive in 20% (95% CI: 11–33) of samples, showing genotype discrepancies with cervical tests. Semen treatment could not completely eliminate the virus in positive samples but reduced the positivity to one‐third. No significant differences in semen and embryological variables, clinical pregnancy and live birth rates, neonatal and obstetrics outcomes were observed in subjects with positivity in semen or cervix compared to respective negative groups. Cumulative live birth rates per oocyte retrieval in couples where both partners were negative or both were positive did not differ, being 37% (95% CI: 28%–47%) and 44% (95% CI: 19–73), respectively. In conclusion, HPV testing should not be considered a prerequisite for accessing ART treatments. Robust inferences for natural fertility cannot be made using our findings, as the ART setting does not fully reflect natural conditions.