The main goal of this study was to investigate, through a biomolecular study, the correlation between papillomatosis of the vulvar vestibule and human papillomavirus (HPV) infection, as well as to establish the necessity of treatment. A total of 44 female adolescents between 12 and 18 years of age were selected through a prospective study with a confirmed diagnosis of papillomatosis of the vulvar vestibule. Vulvar biopsies were obtained for the histological and biomolecular detection of HPV DNA through polymerase chain reaction (PCR). Twenty (45%) adolescents were virgins (group A), the other 24 (55%) were sexually active. The virgin adolescents (group A) and 12 sexually active adolescents (group B) did not show cytological and/or colposcopic alteration, suggesting infection by HPV either on the cervix or vagina. These were compared with 12 other sexually active adolescents who showed cervicovaginal infection caused by HPV (group C). Fisher exact test was applied for statistical analysis of the results, considering alpha equal or less than 0.05. There was no statistically significant difference in relation to HPV DNA through PCR among virgin and sexually active adolescents in group B, however, both differed from those in group C (A + B C: p = 0.048*). The histological study did not reveal evident signs of infection caused by HPV on vestibular papillae, besides perinuclear halos. HPV DNA was detected on vestibular papillae in 27%. Our results confirmed a scarce correlation between vestibular papillae and HPV. Thus, we consider papillomatosis of the vulvar vestibule, in most cases, to be equivalent to physiological papillomatosis and, therefore, should not be treated.