Objectives: To investigate the effects of combined irrigation of EDTA and NaOCl on the remaining bacteria in infected canals after enlarged with ProFile .04 taper no. 30 to 60. Histological and SEM evaluation of the canals were also performed. Methods: Sixty mandibular molars were inoculated with E. faecalis. Teeth were then divided into 3 groups. Group I: irrigated with NSS (Control group), group II: NaOCl and group III: EDTA alternated with NaOCl. After coronal flaring with ProTaper files, MB and ML canals were prepared with ProFile .04 taper no. 30 to 60. Bacterial cells were collected from MB canals after finished with each file sized. Teeth from group II and III were prepared for histological evaluation. The longitudinal sections were stained with Brown and Brenn for bacterial observation. Structures of dentinal tubules were investigated by SEM. Results: The remaining bacteria from each file size were not statistically significant differences between group II and group III (p>0.05). Bacterial reductions was significantly decreased from no. 30 to 35 in all groups (p<0.0001). The light microscopic study found that the pulpal walls of group II were smooth while in group III found irregular in appearance with minimal remaining bacteria in dentinal tubules. SEM study showed the smear layer was still intact in group II while in group III, the smear layer was completely removed and excessive erosion of the intertubular and peritubular dentin was seen. Conclusions: The use of EDTA alternated with NaOCl and NaOCl alone have the same result in bacterial reduction but both irrigating regimens showed better result than using NSS. Apical enlargement up to size 35 significantly reduced the amount of bacteria. Histological and SEM studies revealed that there were irregularity along canal walls and excessive erosion of the internal tubular and peritubular dentin when using EDTA alternated with NaOCl.