Objective: The aim of this study is to evaluate and compare dentinal tubule penetration of various root canal sealers obturated after the application of different irrigation activation procedures by a laser scanning confocal microscope. Methods: A total of 150 extracted human permanent mandibular premolar single-rooted teeth were selected and randomly divided into 3 main groups according to the sealer type (n = 50) as AH Plus, MTA Fillapex, and Sealapex. Each main group was randomly subdivided into 5 subgroups according to the irrigation activation protocols (n = 10): Potassium–titanyl–phosphate laser irradiation, conventional needle, intra-kit, sonic irrigation, and ultrasonic irrigation procedures. After the activation procedures, the root canals were obturated with AH Plus, MTA Fillapex, and Sealapex mixed with 0.1% fluorescent rhodamine B isothiocyanate. Specimens were sectioned at 3, 6, and 9 mm from the apex. All sections were examined under a confocal laser scanning micro- scope to calculate the dentinal tubule penetration area. Results: Data were analyzed using a 3-way analysis of variance and Tukey’s post hoc tests (P = .05). Sealapex indicated a statistically lesser penetration than the other group (P < .05), MTA Fillapex ensured deeper penetration than AH Plus (P < .05). Ultrasonic irrigation provided sig- nificantly deeper penetration than other activation procedures (P < .05). The statistically high- est percentage and the maximum depth of sealer penetration were shown in coronal third for all groups (P < .05). Conclusion: The selection of root canal sealer, irrigation activation procedures, and root canal region plays a crucial role on the dentinal tubule penetration. AH Plus and MTA Fillapex applied with ultrasonic irrigation could achieve deeper sealer penetration in dentinal tubules.