Purpose To demonstrate and understand the association of HPV infection and biofilm formation. Methods The study consisted of cervicovaginal samples of 72 women who were evaluated at the colposcopy unit. Papanicolaou staining was used for cytological examination while "Crystal Violet Binding" assay was performed to detect biofilm formation. Results HPV-DNA was positive in 55.5% (n = 40) of the patients. The biofilm formation rate was statistically significantly higher in the HPV-positive women (45%) compared to HPV-negative women (21.9%) (P < 0.05). There was a statistically significant relationship between the presence of single HPV and "high-risk HPV" types and biofilm formation (P < 0.05). Biofilm formation was found in 80% of women with abnormal smear demonstrating atypical epithelial cells (P < 0.05). Conclusion Biofilm formation is more frequent at the cervicovaginal microbiota of patients with HPV infection. This finding is especially important in cases with atypical epithelial cells at their cervicovaginal smears.