Enterococcus faecalis is the most predominant bacteria in teeth with failed root canal therapy and is found to survive harsh conditions prevailing in the root canals of endodontically treated teeth. This study aims to investigate the interaction between E. faecalis and root canal dentine substrate. Towards this end, tooth specimens were prepared and divided into two groups. The tooth specimens in group 1 were incubated with E. faecalis for periods of 2-, 4-, and 6-week intervals and the chemical composition of the biofilm was determined using X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy. The tooth specimens in group 2 were incubated with E. faecalis for a period of 6 weeks and the topography and ultrastructure of the biofilm were examined using scanning electron microscopy (SEM), light microscopy, and laser confocal scanning microscopy. The sediments formed from the bacterial interaction on the dentine (in group 1) were also examined by SEM and FTIR. These experiments highlighted different stages in the interaction of E. faecalis with root canal dentine. Further, a bacterial-induced apatite reprecipitation on mature biofilm was also observed. This ability of E. faecalis to form such calcified biofilm on root canal dentine may be a factor that contributes to their persistence after endodontic treatment.