Introduction: the study aimed to evaluate Enterococcus Faecalis colonization in the pulp chamber in pulpotomized extracted human teeth filled by different pulpotomy base materials (PBMs), using confocal laser scanning microscopy (CLSM). Study design: Cavity preparations were made in 70 extracted primary molars. The pulp chambers were filled using either Intermediate restorative material (IRM), Mineral Trioxide Aggregate (MTA) or Glass ionomer (GI). Twenty-five teeth served controls. The specimens were sterilized, and coronally filled with bacterial suspension for 21 days. The specimens were cut through the furcation area, stained using LIVE/DEAD BacLight Bacterial Viability Kit and evaluated using CLSM. Results: The extent of fluorescent staining was larger in the GI group, compared to the IRM and MTA groups, and larger in the IRM group compared to the MTA group (P<0.05). The minimal and maximal bacterial penetration depths into the dentinal tubules were 55 and 695μm, respectively (mean 310μm), without differences between the materials (GI, IRM, MTA, p>0.05). The ratio of live bacteria to dead bacteria within the evaluated areas was higher in the GI group compared to the IRM and the MTA groups, and higher in the IRM group compared to the MTA group (P<0.05). There were no differences between the mesial, distal and apical parts in any of the evaluations (p>0.05). Conclusions: bacteria colonize the interface between the PBM and dentin and penetrate deeply into the dentinal tubules. The extent and the vitality of the colonized bacteria may be affected by the type of PBM.