Aim To compare the removal of root fillings in extracted teeth using the ProTaper Universal Retreatment system (PTUR) followed by a supplementary preparation with the XP-Endo Finisher file. Methodology The mesiobuccal root canals of 30 extracted mandibular first molars were instrumented with ProTaper Universal NiTi files up to F2 and filled with one of the following sealers using a single-cone technique (n = 10): AH Plus, NeoMTA Plus and EndoSequence BC. The root fillings were removed using the PTUR system with additional apical preparation using ProTaper F2 and F3 files. Then, an additional preparation with an XP-Endo Finisher file was performed. The samples were scanned using micro-CT before and after retreatment and again after the use of the XP-Endo Finisher to assess the volume of remaining filling material. Data were analysed by Kruskal-Wallis and Friedman's two-way analysis of variance tests with Bonferroni correction. Results No significant difference in the initial filling volume was observed amongst the groups (P > 0.05). The amount of filling materials in each group decreased significantly after retreatment with rotary files (P < 0.05). After initial retreatment, the volume of the remaining filling amongst the groups was similar (P > 0.05). Additional preparation with the XP-Endo Finisher improved the removal of filling materials regardless of the sealer type (P < 0.05). Conclusions In the mesiobuccal root canals of extracted mandibular first molars, additional preparation using an XP-Endo Finisher file, after using the PTUR system and F3 ProTaper instruments, enhanced the removal of filling materials from root canals.