Most oral microorganisms exist as biofilms which initiate formation via the attachment of an early colonizer to host proteins on the tooth surface. Fusobacterium nucleatum act as a bridge between early and late colonizers. Dental biofilms eventually comprise dental pathogens such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. To evaluate the effects of mutual interactions between oral bacteria on the growth of biofilms, periodontopathogens were co-cultured with a 0.4 μm barrier. Streptococcus gordonii inhibited the growth of F. nucleatum and periodontopathogens. However, F. nucleatum, P. gingivalis and T. denticola activated the growth of other bacteria. A co-culture system of early and late colonizers could be a useful tool to further understand bacterial interactions during the development of dental biofilm.