The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate (MTA) was studied by scanning electron microscopy (SEM). Single-rooted human premolars (n560) were instrumented to an apical size #50/0.06 using ProFile and treated as follows:Group 1 (n510) was filled with phosphate buffered saline (PBS);Group 2 (n510) was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS;Group 3 (n520) was obturated orthograde with a paste of OrthoMTA (BioMTA, Seoul, Korea) and PBS;and Group 4 (n520) was incubated with E. faecalis for 3 weeks and then obturated with OrthoMTA–PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material (IRM), and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoMTA-filled roots (Groups 3 and 4). Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots (Group 4). Therefore, the orthograde obturation of root canals with OrthoMTA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization.