ABSTRACTHuman oral bacterial pathogens grow in attached multispecies biofilm communities. Unattached cells are quickly removed by swallowing. Therefore, surface attachment is essential for growth, and we investigated multispecies community interactions resulting in mutualistic growth on saliva as the sole nutritional source. We used two model systems, saliva-coated transferable solid-phase polystyrene pegs (peg biofilms) and flow cells with saliva-coated glass surfaces. Fluorescent antibody staining and image analysis were used to quantify the biomass in flow cells, and quantitative real-time PCR with species-specific primers was used to quantify the biomass in peg biofilms. Veillonellasp. strain PK1910, Aggregatibacter actinomycetemcomitansJP2, and Fusobacterium nucleatumATCC 10953 were unable to grow as single species in flow cells. Only A. actinomycetemcomitansgrew after 36 h when peg biofilms remained submerged in saliva from the time of inoculation. Mixed-species coaggregates were used for two- and three-species inoculation. The biomass in two-species biofilms increased in both systems when Veillonellasp. strain PK1910 was present as one of the partners. Enhanced growth of all strains was observed in three-species biofilms in flow cells. Interestingly, in flow cells F. nucleatumand A. actinomycetemcomitansexhibited mutualism, and, although F. nucleatumwas unable to grow with either of the other species in the peg system, F. nucleatumstimulated the growth of Veillonellasp. and together these two organisms increased the total biomass of A. actinomycetemcomitansin three-species peg biofilms. We propose that mutualistic two-species and multispecies oral biofilm communities form in vivo and that mutualism between commensal veillonellae and late colonizing pathogens, such as aggregatibacteria, contributes to the development of periodontal disease.