An understanding of biofilm behavior of periodontopathic bacteria is key to the development of effective oral therapies. We hypothesized that interspecies bacterial aggregates play an important role in anaerobic biofilm establishment and proliferation, and contribute to the survivability of the biofilm against therapeutic agents. The system developed in this study assessed a multi-species (Streptococcus gordonii, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum) biofilm formation under anaerobic and flowing conditions with the use of an in situ image analysis system. The biofilm was comprised of a base film of non-aggregated cells and complex interspecies aggregates that formed in the planktonic phase which rapidly colonized the surface, reaching 58 +/- 9% and 65 +/- 11.8% coverage by 5 and 24 hrs, respectively. Upon SDS (0.1%) treatment of a 24-hour biofilm, substantial detachment (55 +/- 14%, P < 0.05) of the aggregates was observed, while the base film bacteria remained attached but non-viable. Rapid re-establishment of the biofilm occurred via attachment of viable planktonic aggregates.