Chronic periodontitis is an inflammatory, bacterial biofilm-associated disease resulting in destruction of the tooth’s supporting tissues. The imminent progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Living in a complex oral polymicrobial community, these two bacterial species display close association via physical interaction and metabolic cooperativity in the biosynthesis and cross-feeding of growth substrates. These interspecies interactions result in the coordination of their physiological activities, some of which exhibit complementary effects in enhancing their growth and virulence factors. We have shown that in the presence of T. denticola, P. gingivalis produces free glycine by proteolytic release of peptide-bound glycine, which acts as an important nutrient source for T. denticola. This study aims to demonstrate the synergistic interactions between T. denticola and P. gingivalis in biofilm development and characterise the stimulatory factor(s) release by T. denticola that enable these metabolic interactions. Flow cell system and fluorescent in situ hybridisation using species-specific probes were employed to compare the monospecies and dual-species biofilm formation of P. gingivalis W50 and T. denticola ATCC 35405. Synergism in biofilm development was observed as an increase in the dual-species biofilm biomass and average thickness. Characterization of T. denticola stimulatory factor(s) for the production of P. gingivalis glycine was performed using different fractionation methods and indirect measurement of the stimulatory effects using a Glycine ELISA kit and liquid chromatography- triple quadrupole mass spectrometry (LC-QQQ-MS). Initial results showed that the T. denticola stimulatory factor(s) is less than 3 kDa in size and is able to enhance P. gingivalis glycine production. Stimulatory molecules released by T. denticola to enhance metabolic substrate production by P. gingivalis may be one of the mechanisms employed to achieve an enhancement in growth and virulence.