Aim To evaluate the removal of accumulated hard‐tissue debris (AHTD) from the root canal system of mandibular molars by positive and negative pressure irrigation systems, using micro‐CT imaging analysis. Methodology Mandibular molars with a single canal in the distal root and 2 canals connected by an isthmus in the mesial root were matched based on similar morphological dimensions using micro‐CT evaluation and assigned to 2 experimental groups (n = 20 mesial and 10 distal canals), according to the irrigation protocol: apical positive (conventional irrigation) or negative (EndoVac system) pressure. Changes in root canal volume and surface area as well as percentage of uninstrumented canal wall surface and accumulated hard‐tissue debris (AHTD) after canal preparation were compared statistically using the independent sample t‐test and Mann–Whitney U‐test, with the significance level set at 5%. Results Volume, surface area and percentage of static voxels in either mesial or distal root canal systems were not significantly different between groups before or after root canal preparation (P > 0.05). After preparation, AHTD was not observed in the distal canal of both groups. However, in the mesial root canal system, the conventional irrigation group was associated with a significantly higher median percentage of AHTD (11.48%; IQR: 5.9–22.6; range: 1.86–41.98) than the EndoVac group (3.40%; IQR: 1.5–7.3; range: 0.82–12.84) (P < 0.05). Conclusions Neither irrigation protocol succeeded in rendering the mesial canal system free of AHTD; however, apical negative pressure irrigation resulted in lower levels of AHTD than conventional irrigation.