Purpose: This study evaluated the effect of 17% EDTA, 10% citric acid (CA), and 2% chlorhexidine (CHX) activated with a rotary microbrush (CanalBrush) on root dentin microhardness, roughness, and epoxy-based sealer bond strength. Materials and Methods: One hundred sixty single-rooted bovine incisors were instrumented and divided into 8 groups according to treatment: 1. 17% EDTA; 2. 17% EDTA+2% CHX; 3. 10% CA; 4. 10% CA+2% CHX; 5. 17% EDTA with CanalBrush; 6. 17% EDTA+2% CHX with CanalBrush; 7. 10% CA with CanalBrush; and 8. 10% CA+2% CHX with CanalBrush. Ten roots in each group were split into halves and submitted to microhardness and roughness analyses (n = 10). Following endodontic filling with AH Plus sealer, 10 roots in each group underwent push-out bond strength testing (n = 10). Data were analyzed using two-way ANOVA and Tukey's tests (α = 0.05). Results: All groups had similar microhardness values (p > 0.05) which was higher in the apical third than in the middle and cervical thirds (p 0.05). The CanalBrush groups had higher roughness than the no-activation groups (p 0.05), with significantly higher roughness in the cervical third than in the apical third (p 0.05). All groups exhibited similar bond strength (p > 0.05), with the cervical third being higher, followed by the middle and apical thirds (p 0.05). Conclusions: Microbrush activation had a direct impact on dentin roughness and did not influence the dentin microhardness or the retention of epoxy-based sealer to the root canal. Keywords: EDTA, endodontics, epoxy resin based root canal sealer, root canal irrigantsaler