Introduction: The present study evaluated the effect of the source of biofilm bacteria on their susceptibility in dentinal tubules to disinfecting solutions using an infected dentin model. Methods: Infected dentin blocks were prepared. Enterococcus faecalis strains VP3-181 and Gel 31 were introduced into dentinal tubules by centrifugation to form monospecies biofilms, whereas 3 specimens of pooled plaque bacteria collected from different donors were used to grow multi species biofilms in dentin. After 1 and 3 weeks of incubation, the samples were subjected to sterile water, 2% chlorhexidine (CHX), and 2% sodium hypochlorite (NaOCl). After the 3-minute exposure, the proportions of killed bacteria in dentin canals were assessed by viability staining and confocal laser scanning micro-scopy. Results: The proportion of killed bacteria in mature (3 weeks) mono- and multispecies biofilms was lower than in young biofilms (1 week) after treatment (P < .05). 5). E. faecalis Gel 31 biofilms and multi species biofilms were more resistant than VP3-181 biofilms. No differences in the susceptibilities to the disinfecting agents of the 3 multispecies biofilms were detected; 2% NaOCl was more effective against multi species biofilms in dentin than 2% CHX (P < .05), whereas no significant difference was detected between 2% CHX and 2% NaOCl against the E. faecalis strains. Conclusions: Mature mono- and multispecies biofilms in dentinal tubules are more resistant to disinfectants than corresponding young biofilms. The susceptibility of the monospecies E. faecalis dentin biofilm showed strain-related differences, whereas the multispecies biofilms from different donors showed similar susceptibility.