Key bacterial vaginosis (BV)-associated bacteria implicated in biofilm formation include Gardnerella species, Prevotella bivia , and Fannyhessea vaginae . We investigated their spatial organization in the BV biofilm over time from longitudinal vaginal specimens obtained from women with incident BV (iBV) using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH). Heterosexual women with optimal vaginal microbiota self-collected vaginal specimens twice daily for 60 days or until iBV development (Nugent score 7–10 on ≥4 consecutive specimens). Women who developed iBV were matched to healthy controls by age, race, and contraceptive method. Gardnerella spp., P. bivia , and F. vaginae were quantified using PNA-FISH 2 days pre-iBV, the day of iBV, and 2 days post-iBV across five optical layers ( z , z + 2, z + 4, z + 6, and z + 8 μm). Total counts of all three bacterial species were significantly higher on the day of iBV compared to 2 days pre-iBV ( P = 0.011) and remained elevated 2 days post-iBV. Across most layers and time points, pooled mean Gardnerella spp. counts were significantly higher than F. vaginae counts ( P ≤ 0.022-0.0003). On the day of iBV and 2 days post-iBV, pooled mean counts of Gardnerella spp. and F. vaginae progressively increased across most biofilm layers ( P ≤ 0.043-0.0012). Controls had significantly lower counts of Gardnerella spp. and F. vaginae. P. bivia had low counts in all specimens. During the critical time period surrounding iBV, Gardnerella spp. are abundant throughout the developing biofilm and facilitate F. vaginae incorporation at later time points and higher biofilm layers. Additional research, including other Prevotella spp., is needed. IMPORTANCE: Bacterial vaginosis (BV) is the most common vaginal infection in reproductive-age women worldwide with a global prevalence of 30%. Recurrence rates can be up to 60% within 1 year of treatment. While BV is characterized as a polymicrobial biofilm infection, the exact etiology remains unknown. The BV biofilm may persist after antibiotic treatment, possibly due to incomplete eradication by current antimicrobial therapies, contributing to recurrent infection. Data are limited in evaluating the spatial formation of the BV biofilm around the time of incident BV. Providing a better understanding of this critical time period in incident BV pathogenesis is necessary to inform the development of prevention methods aimed at inhibiting biofilm formation and improving long-term treatment outcomes.

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