Background: Dental caries is a microbe and sugar-mediated bioflm-dependent oral disease. Of particular signifcance, 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 signifcance 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 bioflm model to assess how exogenous pathogens could impact the structural and functional characteristics of the indigenous native oral microbiota. Results: Through shotgun whole metagenome sequencing, we observed that saliva-derived bioflm has decreased richness and diversity but increased sugar-related metabolism relative to the planktonic phase. Addition of S. mutans and/or C. albicans to the native microbiome drove signifcant changes in its bacterial composition. In addition, the efect of the exogenous pathogens on microbiome diversity and taxonomic abundances varied depending on the sugar type. While the addition of S. mutans induced a broader efect on Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog abundances with glucose/fructose, S. mutans-C. albicans combination under sucrose conditions triggered unique and specifc changes in microbiota composition/diversity as well as specifc efects on KEGG pathways. Finally, we observed the presence of human epithelial cells within the bioflms via confocal microscopy imaging. Conclusions: Our data revealed that the presence of S. mutans and C. albicans, alone or in combination, as well as the addition of diferent sugars, induced unique alterations in both the composition and functional attributes of the bioflms. In particular, the combination of S. mutans and C. albicans seemed to drive the development (and perhaps the severity) of a dysbiotic/cariogenic oral microbiome. Our work provides a unique and pragmatic bioflm model for investigating the functional microbiome in health and disease as well as developing strategies to modulate the microbiome.