The purpose of the present study was to examine whether bacterial biofilm concerned with bacterial colonization and growth of subgingival plaque. We observed subgingival plaque of 95 severe adult periodontitis-affected teeth by scanning electron microscope. In the coronal one-third zone of subgingival plaque, cocci, rods and others established biofilm by glycocalyx-like structure. In the middle one-third zone, several kinds of bacterial species were coaggregated. Film-like microcolonies were also observed in this zone. We detected filamentous microorganisms appeared to come out from the film-like colonies, and observed holes which microorganisms had been passed through the colonies. In the apical one-third zone, several kinds of bacterial species aggregated by glycocalyx-like structures, which comprised netlike and mucoidal structure. In the “plaque-free zone”, two types of bacterial colonization were observed : bacterial cell wall directly contacted with the root surface, and microorganisms adhered to the root surface mediated by glycocalyx netlike materials. In this zone, five periodontal disease-associated bacteria, including Porphyromonas gingivalis, were detected using back-scattered electron imaging modes. The results of the present study suggested that bacterial biofilms were partially related to colonization and growth of subgingival plaque.