To investigate how early mono- and dual species biofilms of oral Actinomyces naeslundii and Streptococcus gordonii clinical isolates metabolized glucose in the presence of the complex salivary mucin MUC5B, this study employed NMR-based metabolomics with interpretation of network integration. Untargeted NMR data analysis for metabolite detection and annotation and in silico studies of biochemical relationships between metabolites and cellular processes was performed to study functional differences in the early biofilm metabolomes. Significant differences in metabolite abundance elicited by the presence of MUC5B were detected. The main metabolites that distinguished the biofilms grown with 25% MUC5B with 20 mM glucose (MUC5B+G) from those grown with physiologic buffer with 20 mM glucose (PBS+G) were the unannotated metabolites 68 and 73. In the conditions were they were each present, pyruvate, ethanol and metabolite 134 were significantly more abundant in the presence of MUC5B, while metabolites 84, 97 and sarcosine were significantly less abundant. The pathway enrichment analysis of the metabolites that were differently expressed in early A. naeslundii, S. gordonii and dual species biofilms grown with PBS+G or MUC5B+G showed that pyruvate metabolism was significantly over-represented. Studying the metabolic interactions between commensal members of oral biofilms and modulatory effects of host factors such as glycoproteins in saliva during metabolism of substrates that are potential drivers of dysbiosis, such as glucose, is essential to understand the roles of oral microbial ecosystems in oral health and disease.