Aim To determine and compare the accuracy and efficiency of a dynamic navigation system (DNS) with a freehand (FH) technique when conducting root-end resection in a human cadaver model. Methodology Forty roots in cadaver heads were included and divided into two groups: DNS (n = 20) and FH (n = 20). Cone beam computed tomography (CBCT) scans of all teeth were taken. The drilling path and depth were planned virtually to using the Xguide software (X-Nav Technologies, Lansdale, PA, USA). Osteotomy and root-end resection were done under navigation in the DNS group, and freehand under the dental operating microscope in the FH group. Post-operative CBCTs were taken. Linear deviations, angular deflection, time of operation and number of mishaps were compared with determine the accuracy and efficiency. Shapiro-Wilk, one-way ANOVA and Fisher exact tests were used (P < 0.05).Results Linear deviations, angular deflection and operation time were significantly less in the DNS group (P < 0.05). The number of mishaps was not different between the two groups (P > 0.05). Subgroup analyses revealed that the distance of >5 mm from buccal cortical plate was significantly associated with lower accuracy, increased operation time and greater incidence of mishaps in the FH group (P < 0.05), but not in the DNS group. Conclusions The dynamic navigation system was more accurate and more efficient in root-end resection in a cadaver model than the freehand technique. The distance of the roots from the buccal cortical plate had a significant negative impact on the accuracy and efficacy of the root-end resection procedure when using the freehand technique. The dynamic navigation system has the potential to be a safe and reliable technological addition to endodontic microsurgery.