Background: Laser-assisted endodontic treatment is a suitable instrument for smear layer removal in root canals. Objective: This ex-vivo study evaluated the efficiency of an Er: YAG laser (2940nm) at different pulse energy levels to remove smear layer with or without chelators from the middle and apical third of mesial roots. Methods: This study evaluated the efficiency of an Er: YAG laser (2940nm) at different pulse energy levels to remove smear layer with or without chelators from the middle and apical third of mesial roots. 48 mesial root canals of first mandibular molars were divided into 4 groups of 12 teeth each. Each group consisted of 2 subgroups each (A and B), regarding the irrigation protocol. In subgroup A, teeth were rinsed only by distilled water whereas in subgroup B, teeth were rinsed by 5ml 17% EDTA for 60sec, 5ml 5% NaOCL and 5ml distilled water. Four different pulse energy values were tested, namely 30mJ, 50mJ, 70mJ, 80mJ in order to irradiate the roots in Group 1, Group 2, Group 3 and Group 4 respectively. Control groups (n=4) was instrumented and rinsed as experimental groups (subgroup A & B) but not irradiated. Teeth were sectioned longitudinally and observed under SEM. Results: Results were statistically analyzed with the Kruskal-Wallis Test. There is statistically significant difference between groups irrigated with chelators and groups with distilled water before laser irradiation in the apical third. Group 1B (0.75W) showed statistically significant outcome in apical part. The results showed no statistically difference between subgroup B and control group. Conclusion: The presence of chelating factor may play an important role in the laser mechanism of smear layer removal from apical part of narrow and curved root canals.