© 2022 Wolters Kluwer Medknow Publications. All rights reserved.Background: The whole root canal disinfection is crucial in terms of long-term endodontic success. In this context, applying medicaments following effective irrigation activation procedures becomes an important point. Aims: The purpose of this study was to evaluate and compare the efficacy of various irrigation activation techniques on non-infected dentinal tubule penetration of calcium hydroxide (CH), double (DAP), and triple (TAP) antibiotic pastes. Materials and Methods: A total of 180 extracted human permanent mandibular premolar single-rooted teeth were selected and randomly divided into a control group and four main groups according to the irrigation activation procedures (n = 36) as KTP laser irradiation, conventional needle, NaviTip FX, sonic (SI) and ultrasonic activation (UI) procedures. Each group was randomly subdivided into three subgroups according to the medicament type (n = 12); CH, DAP, and TAP. After the activation procedures, the root canals were filled with CH, TAP, and DAP mixed with 0.1% fluorescent rhodamine B isothiocyanate. Specimens were sectioned at 2, 5, and 8 mm from the apex, and all the sections were examined under a confocal laser scanning microscope to calculate the dentinal tubule penetration. Data were analyzed using a three-way analysis of variance and Tukey's post hoc tests (P = 0.05). Results: TAP provided a statistically significant greater penetration than the other groups (P < 0.05). UI provided a statistically significant higher dentinal tubules penetration area than other activation procedures (P < 0.05). Conclusion: Medicament penetration depends on the type of medicament, activation procedures, and root canal level. The TAP may be preferred following the ultrasonic activation in terms of long term root canal treatment success.