Background <p>This study aimed to evaluate, using confocal laser scanning microscopy (CLSM), the dentin tubule penetration of a calcium silicate–based root canal sealer after removal of calcium hydroxide (Ca(OH)₂) or N-acetylcysteine (NAC) intracanal medicaments with needle irrigation or EDDY sonic activation.</p> Methods <p>Root canals of 128 extracted single-rooted mandibular premolars were prepared with ProTaper Next. Canals received Ca(OH)₂ (Kalsin) or (NAC) for 7 days. Medicaments were removed using conventional needle irrigation or EDDY sonic activation. Specimens were obturated with a calcium silicate–based sealer Bioserra using the single-cone technique. CLSM quantified residual medicament and sealer penetration into dentin tubules. Statistical analyses were performed using the Mann–Whitney U and Kruskal–Wallis tests, with the significance level set at <i>p</i> &lt; 0.05.</p> Results <p>Neither the type of intracanal medicament nor the activation technique significantly affected the residual medicament amount (<i>p</i> &gt; 0.05). However, the type of medicament significantly influenced sealer penetration, with NAC showing greater dentin tubule penetration than Ca(OH)₂ (<i>p</i> &lt; 0.05). The activation technique did not significantly influence sealer penetration (<i>p</i> &gt; 0.05). Sealer penetration was highest in the coronal third and lowest in the apical third (<i>p</i> &lt; 0.05).</p> Conclusion <p>Within the limitations of this in vitro study, NAC enhanced the dentin tubule penetration of a calcium silicate–based sealer compared with Ca(OH)₂. The activation technique did not affect sealer penetration or residual medicament.</p>