Background <p>The human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide. Only a small percentage of high-risk (HR) HPV infections progress to cervical precancer and cancer. Recent research indicates the potential association between the variation of vaginal microbiota and the acquisition and persistence of human papillomavirus (HPV) infection. However, the association of STIs with HPV cervical infection and cervicovaginal lesions has not yet been fully elucidated. The aim of this study was to assess how <i>G. vaginalis</i> is associated with HPV infection and cervical dysplasia, and the beneficial role of <i>Lactobacillus</i>.</p> Methods <p>A total of 723 women aged between 17 and 79 years were studied. A complete cervico-vaginal swab for a cervico-vaginal cytological examination (Pap test) and the simultaneous identification, by Real-Time PCR, of the presence of HR-HPV and other STI pathogens, was conducted.</p> Results <p>The HPV positive women were 37.2%, with a prevalence significantly different in the various age groups. The most frequent genotype was HPV-16, while <i>G. vaginalis</i> was found in 369 women, in which 51.8% of cases occurred together with HPV-HR, highlighting a significant association between the two infections (<i>p</i> &lt; 0.001). Moreover, a marked reduction in HR-HPV infection was observed in the presence of <i>Lactobacillus</i>. (<i>p</i> &lt; 0.001). All-grade lesions were significantly associated with <i>G. vaginalis</i> and detected in 65.4% of <i>G. vaginalis</i>-positive samples (<i>p</i> &lt; 0.01).</p> Conclusions <p>The results of this study strengthen the hypothesis of the association between HPV infections and microbiota in cervical lesions.</p> Clinical trial number <p>Not applicable.</p>