Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. The Gram-negative Bacteroidetes species Porphyromonas gingivalis and Tannerella forsythia and the Spirochaete Treponema denticola have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. Little is known of T. denticola factors important for polymicrobial biofilm formation. The aims of this study were initially to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia. Then to determine the roles of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant and the role of T. denticola motility using a flagellar mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms in this system. A strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola. The T. denticola nonmotile mutant formed structurally different polymicrobial biofilms with P. gingivalis that lacked the defined intracolonial channels of the wild-type biofilms. These data support the synergistic pathogenesis between these species that occurs in a murine model of disease.