ObjectiveThis study used high-throughput amplicon sequencing to examine the impact of long-term continuous fluoride treatment on the dental plaque microbiota of children aged 8 to 9 with mixed dentition. DesignThe study population consisted of twenty 8–9-year-old children with dental caries. Topical application of fluoride-varnish was weekly administered for one month to all subjects. Clinical indicators and anthropological data, such as the caries index (DMFT and dmft), were documented for every participant at baseline. A baseline assessment and a month after the fluoride varnish treatment were conducted for the salivary pH level and the Patient-Hygiene-Performance (PHP) index. Following application of the fluoride varnish, plaque samples were obtained both one month later and before (baseline) and were then used for 16S rRNA gene-based Next Generation Sequencing. ResultsThe results showed significant differences in the community composition structure (p < 0.01). Notable caries-associated pathogens in the dental plaque microbiome were depleted whilst health associated phylum Proteobacteria was increased in the abundance following fluoride-varnish application. In children with mixed dentition, this study found that after one month of fluoride-varnish treatment, there was a significant decrease in the prevalence of the dominant pathogenic genera, Fusobacterium, Porphyromonas, Capnocytophaga, Neisseria, and Leptrotrichia, along with an increase in certain genera related to healthy oral condition, mostly from the phylum Proteobacteria, such as Areinmonas, Pseudoxanthomonas, and Luteimonas. ConclusionsFluoride-varnish application may shift the community level microecology from dysbiosis to eubiosis. Moreover, application of fluoride-varnish with weekly intervals for one month reduced the caries-causing bacteria while enriching the rise of unique, ubiquitous genera primarily belonging to the Proteobacteria, which may plaque a defensive role against progression of caries. Furthermore, a rising pH level towards neutrality (pH 7) indicated a healthier oral environment following the application of fluoride varnish.