BackgroundDental caries is a microbe and sugar-mediated biofilm-dependent oral disease. Of particular significance, a virulent type of dental caries, known as severe early childhood caries (S-ECC), is characterized by the synergistic polymicrobial interaction between the cariogenic bacterium,Streptococcus mutans, and an opportunistic fungal pathogen,Candida albicans. Although cross-sectional studies reveal their important roles in caries development, these exhibit limitations in determining the significance of these microbial interactions in the pathogenesis of the disease. Thus, it remains unclear the mechanism(s) through which the cross-kingdom interaction modulates the composition of the plaque microbiome. Here, we employed a novel ex vivo saliva-derived microcosm biofilm model to assess how exogenous pathogens could impact the structural and functional characteristics of the indigenous native oral microbiota.ResultsThrough shotgun whole metagenome sequencing, we observed that saliva-derived biofilm has decreased richness and diversity but increased sugar-related metabolism relative to the planktonic phase. Addition ofS. mutansand/orC. albicansto the native microbiome drove significant changes in its bacterial composition. In addition, the effect of the exogenous pathogens on microbiome diversity and taxonomic abundances varied depending on the sugar type. While the addition ofS. mutansinduced a broader effect on Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog abundances with glucose/fructose,S. mutans-C. albicanscombination under sucrose conditions triggered unique and specific changes in microbiota composition/diversity as well as specific effects on KEGG pathways. Finally, we observed the presence of human epithelial cells within the biofilms via confocal microscopy imaging.ConclusionsOur data revealed that the presence ofS. mutansandC. albicans, alone or in combination, as well as the addition of different sugars, induced unique alterations in both the composition and functional attributes of the biofilms. In particular, the combination ofS. mutansandC. albicansseemed to drive the development (and perhaps the severity) of a dysbiotic/cariogenic oral microbiome. Our work provides a unique and pragmatic biofilm model for investigating the functional microbiome in health and disease as well as developing strategies to modulate the microbiome.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s40168-023-01561-7.