Endodontic treatment is aimed at eliminating infection and preventing bacterial regrowth in the root canal and dentinal tubules. In the present study the ability of two dentin adhesives to prevent bacterial penetration and subsequent proliferation in dentinal tubules was evaluated. Cylindrical root specimens prepared from freshly extracted bovine teeth were used in an in vitro model of dentinal tubule infection. After removal of the smear layer the intracanal dentinal tubules of the specimens were acid-etched and treated with either Gluma or EBS. Untreated specimens served as controls. Specimens were infected with Enterococcus faecalis and incubated in Brain Heart Infusion for 21 days. Powder dentin samples obtained from within the canal lumina, using ISO 025 to 033 burs, were examined for the presence of vital bacteria by inoculating on agar plates and counting colony-forming units. A significant difference was found between the experimental groups and the untreated group. After application with Gluma specimens showed the least viable bacteria in dentinal tubules. Data suggested that dentin adhesives reduced bacterial invasion into dentin and therefore have a potential role in endodontic treatment.