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Background: Only recently the concept of prebiotics has been introduced in oral health. Few potential oral prebiotics have already been identified in dual species competition assays, showing a stimulatory effect on beneficial bacteria and by this suppressing the outgrowth of pathogenic species. This study aimed to validate the effect of previously identified potential prebiotic substrates on multispecies cultures by shifting the biofilm composition towards a more beneficial species dominated microbiota. Methods: A chemostat culture containing 14 model oral bacterial species was used to grow biofilms for 24 hours which subsequently were treated with prebiotic solutions three times a day for 3 consecutive days. Further the influence of environmental factors such as pH, nutrient availability, oxygen concentration and prebiotic dose on the efficacy of the prebiotic substances was investigated. Results: Three potential prebiotic substrates N-acetyl-D-mannosamine, succinic acid and Met-Pro were able to bring the beneficial proportion to > 95%. While the pH of the prebiotic solution did not have an influence on the prebiotic effect, the interplay of nutrient availability, oxygen concentration and prebiotic treatment resulted in significant changes of the microbial composition identifying N-acetyl-D-mannosamine as the most promising oral prebiotic substrate. Showing a clear dose dependent effect, concentrations of N-acetyl-D-mannosamine of 1.0 and 1.5 M resulted in a biofilm composition of 97% beneficial species. Conclusion: Introducing the prebiotic concept in oral health might reveal a valid approach for treatment and prevention of oral diseases and promote oral health.