Aim To compare canal and isthmus debris debridement efficacies of the manual dynamic irrigation (MDI) and apical negative pressure (ANP) techniques in the mesial root of mandibular first molars with narrow isthmi, using a closed canal design. Methodology Micro-computed tomography was employed to select 20 teeth, each containing a narrow isthmus. Each root was sealed at the apex with hot glue and embedded in polyvinylsiloxane to simulate a closed canal system. The teeth were submitted to a standardized instrumentation protocol. Final irrigation was performed with either the MDI or the ANP technique using the EndoVac system (N = 10). Masson trichrome-stained sections were prepared from completely demineralized roots at 10 canal levels between 1 and 2.8 mm of the anatomical apices. Areas occupied by canals and isthmus of each root and debris in the corresponding regions were digitized by the NIH Image J software and statistically analysed using two-way repeated measures ANOVA. Results For the instrumented canals, there were no differences between the two groups (P = 0.131) in the area occupied by debris at all canal levels (P = 0.343). Conversely, for the isthmus, less debris was found in the ANP group (P < 0.001) but no differences were seen in each group with respect to the 10 canal levels (P = 0.352). Conclusion Neither technique completely removed debris from the isthmus regions. However, the EndoVac system, which encompasses the ANP concept, removed considerably more debris from narrow isthmi in mandibular mesial roots.