Introduction: The purpose of the present study was to determine the effect of 17% Ethylene, di-amine, tetra-acetic acid (EDTA), 6% phosphoric acid and Erbium: Yttrium, Aluminum, Garnet (Er:YAG) Laser in removing the smear layer by scanning electron microscopy.Methods: In this study, 80 single-rooted human teeth were selected. Instrumentation was done by use of hand files and step-back technique up to file #40 at apical and file #80 at coronal area. During instrumentation, 1ml of 5.25% sodium hypochlorite (NaOCl) was used as irrigation between each file. The teeth were randomly divided into four groups with different methods of smear removal. 17% Ethylene, di-amine, tetra-acetic acid was used in group 1, 6% Phosphoric acid in group 2, Erbium: Yttrium, Aluminum, Garnet laser in group 3, and no intervention in group 4 (as control). Roots were then longitudinally sectioned and prepared for scanning electron micrograph in cervical, middle and apical areas. Data were analyzed by Kruskal-Walis, Mann-Whitney, Friedman and Wilcoxson tests (P<0.05).Results: The results showed a significant difference in smear layer removal between three methods in favor of the EDTA (P<0.001) and Phosphoric groups (P<0.001) with no significant difference between them (P=0.49). Although Er: YAG laser showed some limited ability to remove the smear layer, the effectiveness was not significantly different from the control group (P=0.157).Conclusion: Based on the findings of this study, EDTA and Phosphoric acid were effective methods to remove smear layer from the root canal walls but Er: YAG laser showed less efficacy compared to the other experimental groups.