Background: Apicoectomy and retrograde filling is indicated when conventional endodontic treatment is impossible or failed to achieve apical seal. The aim of this study was to evaluate the effect of ER: YAG laser on apical microleakage. Materials and Methods: Sixty extracted single-rooted teeth were used in this study. The roots were divided into six groups. Group 1: apicoectomy by fissure bur, and apical cavities prepared by round bur, then cavities were filled with MTA. Group 2: the roots preparations and fillings were the same as group 1, then the apical areas were treated by Er:YAG Laser. Group 3: apicoectomy by fissure bur, and apical cavities prepared by ultrasound retrotip and cavities were filled with MTA. Group 4: the roots preparations and fillings were the same as group 3, then the apical areas were treated by Er:YAG Laser. Group 5: the roots obturation with gutta percha, then the apices were resected using Er:YAG Laser. Group 6: the roots apices were resected with Er:YAG Laser, then the canals were obturates with gutta percha. Apical microleakage was measured by methylene blue dye penetration technique. Results: Significant difference between the groups. Group 1 shows the best apical seal, while group 5 shows the worse apical sealing ability. Conclusion: Apicoectomy by bur is better than apicoectomy by laser, and apical cavity prepared by bur is better than prepared by US. The use of ER: YAG laser in apicoectomy is preferred to be done before obturation of the root canal with gutta percha.