Background: Cervical intraepithelial lesions, and subsequently cervical cancer, are significantly caused by persistent infection with high-risk human papillomavirus (HrHPV) types. Lactobacillus-rich vaginal microbiota may protect against HPV, according to recent studies on HPV persistence and clearance. The goal of this study was to determine the effects of probiotic vaginal microbiota regulation on cervical cytology and HPV persistence in women with HPV infection and/or preinvasive cervical lesions. Methods: This study retrospectively examined 142 women who had positive HPV tests. 67 participants received vaginal probiotic therapy (treatment group), while 75 were managed conservatively (control group). Cervical cytology and HPV genotyping were conducted at baseline and 6th month follow-up in all patients. Probiotic strains included Lactobacillus (L.) regenerans, L. helveticus, L. rhamnosus, L. salivarius, and Bifidobacterium (B.) longum. Results: Baseline characteristics showed no significant differences between groups. In 65.7% of the treatment group and 73.3% of the control group, cytological improvement (from ≥Atypical squamous cells of undetermined significance (ASC-US) to normal) was observed (for all p > 0.05). HPV-16 clearance was observed in 47.8% of the treatment group and 55.6% of the control group. There was little variation in HrHPV clearance rates (47.5% versus 48.3%). Only age significantly correlated with persistent HrHPV infection in multivariate analysis (p = 0.020). Conclusions: Cytological improvement and HPV clearance showed a non-significant trend with vaginal probiotic treatment. Age is a significant factor affecting the persistence of HrHPV. Longer follow-up in further randomized controlled studies is necessary to validate the potential benefits of microbiota-targeted therapies.