Bacterial invasion of dentinal tubules and the clinical consequences have been recognized for over a century. However, while many components of the infected dentinal tubule microflora have been identified, it is likely that there are etiological agents involved in endodontic infections that have not yet been recognized. Bacterial invasion of coronal dentinal tubules occurs when the dentine is exposed to the oral environment and of radicular dentinal tubules subsequent to infection of the root canal system or as a consequence of periodontal disease. The content and architecture of a dentinal tubule can influence bacterial invasion, with tubule patency being important. This can account for regional variations in bacterial invasion and is particularly seen with dentinal sclerosis, where more advanced sclerotic changes in apical radicular tubules, especially in elderly individuals, limit bacterial invasion in this area. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria are involved in invasion of dentinal tubules. Gram-positive organisms dominate the tubule microflora in both carious and non-carious dentine. The relatively high numbers of obligate anaerobes present, such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp., suggests that the environment favors the growth of these bacteria. Gram-negative obligate anaerobic rods, e.g. Porphyromonas spp., are less frequently recovered; however, with time, fastidious obligately anaerobic bacteria become established as principal components of the microflora and can be found within the deep dentine layers. In the early stages of infection, Gram-positive bacteria dominate the microflora. The identification of adhesins that mediate these initial interactions of bacteria with dentine is important for understanding the development of tubule infection and in designing adhesion-blocking compounds. Recent evidence suggests that streptococci and enterococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra-tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate invasion of dentine by select bacteria. It is important therefore that the mechanisms of invasion and inter-bacterial adhesion are understood to assist development of novel control strategies.