To evaluate and compare the effect of calcium hydroxide [Ca(OH)2] and 2-hydroxyisocaproic acid (HICA) on the microhardness of root dentine. Fifty-one matured maxillary central incisors with straight root and type I canal configuration were decoronated to a standardized length of 16mm. The root canals were cleaned and shaped using rotary instruments up to size F5. The tooth samples were then randomly assigned into three groups (n = 17) based on the intracanal medicament placed. Group A: control group with no intracanal medicament, Group B: root canals placed with Ca(OH)2, and Group C: root canals placed with HICA. After 1week, the intracanal medicaments placed within the root canals were removed and the canals were dried. Subsequently, the specimens were split longitudinally into two halves and subjected to the Vickers microhardness test with indentations made at the coronal, middle, and apical-third root regions. The data were analyzed using one-way ANOVA and Tukey's post hoc tests. The control group showed significantly highest microhardness value (P < 0.001), followed by HICA and finally, Ca(OH)2 at the coronal, middle, and apical-third root regions, respectively. Nonetheless, there were no statistically significant differences in microhardness values across different root regions within each group. HICA was found to have a significantly lesser reduction in root dentine microhardness which could be a potential alternative to Ca(OH)2. Nevertheless, future studies are warranted to verify the present outcomes in the clinical setting.