Purpose <p>Limited research has explored the impact of retreatment on dentinal microcracks in root canals filled with bioceramic (BC) sealer. The aim of the present study was to evaluate and compare microcrack formation during the removal of BC sealer using the XP-endo Retreatment (XPER) and ProTaper Universal Retreatment (PTUR) systems, employing micro-computed tomography (micro-CT) for detailed analysis.</p> Methods <p>Forty human mandibular premolars with single roots were selected, cleaned, shaped, and obturated with gutta-percha and TotalFill BC sealer using the single-cone technique. After random assignment into Group 1 (PTUR system) and Group 2 (XPER system), pre-treatment micro-CT scans were done. Post-retreatment micro-CT scans were then obtained. Microcracks were quantified before and after retreatment, and statistical analysis was done using Wilcoxon Signed-Ranks, Kruskal–Wallis, and Mann–Whitney U tests (<i>p</i> ≤ 0.05).</p> Results <p>Insignificant differences in microcrack formation were observed between the XPER and PTUR groups. While the PTUR system showed a 15% increase in microcrack formation, this change was insignificant. In contrast, after retreatment, the XPER system demonstrated a significant 45% increase in microcracks. This increase was consistent across all root levels, with the middle third showing a significantly higher number of microcracks than the apical third. Most microcracks result from the propagation of pre-existing cracks.</p> Conclusion <p>The XPER system resulted in more microcracks than the PTUR system, although the difference was not statistically significant. Most microcracks observed after retreatment were propagated from pre-existing cracks, indicating that retreatment primarily exacerbates existing microcracks rather than creating new ones.</p>