Background <p>This study investigated the effect of three intracanal medicaments on the penetration ability of three different root canal sealers using confocal laser scanning microscopy (CLSM).</p> Materials and methods <p>120 extracted single-rooted human teeth were used. The crowns were removed, and each canal was prepared. The teeth were divided into four groups (<i>n</i> = 30): three medicament groups (calcium hydroxide (CH), triple antibiotic paste (TAP), and double antibiotic paste [DAP]) and one control group. 0.1% Rhodamine B was incorporated. After three weeks, the medicaments were removed, and then canals were filled with one of the following sealers: AH Plus, MTA Fillapex, and TotalFill (<i>n</i> = 10). Fluorescein dye was added to the sealers. Horizontal sections (1&#xa0;mm thick) were taken from each canal’s coronal, middle, and apical portions. Samples were then viewed under CLSM. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U tests.</p> Results <p>TotalFill exhibited the highest dentinal tubule penetration values across all root levels and medicament groups, followed by AH Plus and MTA Fillapex. However, these differences were not statistically significant (<i>p</i> &gt; 0.05). All sealers showed greater penetration in the coronal third compared to the apical third.</p> Conclusions <p>Although intracanal medicaments did not significantly affect sealer penetration, TotalFill demonstrated more consistent penetration patterns regardless of the medicament used.</p>