<p>The use of calcium silicate-based root canal sealers has become increasingly widespread. These materials have been shown to interact with dentin and alter its surface characteristics. The ion exchange and moisture interaction occurring during the setting process of the material may lead to changes in dentin microhardness. MTA Fillapex and Well-Root ST are two calcium silicate-based sealers commonly used in clinical practice. The aim of this study was to evaluate the effects of MTA Fillapex and Well-Root ST on dentin microhardness at the coronal, middle, and apical levels of the root. 30 single-rooted premolar teeth were used, and these teeth were first separated into crown and root portions. After determining the canal length, root canal preparation was performed, and the canals were irrigated and dried using paper points. Each tooth was longitudinally sectioned into two halves along the root canal, and the obtained specimens were embedded in acrylic resin. The 30 teeth were divided into two experimental groups and two control groups. After the initial surface microhardness values were measured, the surface microhardness values of the groups treated with root canal sealers were recorded. In both material groups, dentin microhardness values after application were observed to increase compared with the baseline values. Similarly, in the MTA Fillapex group, post-application microhardness values in all regions were found to be significantly higher than the initial values. In intergroup comparisons, Well-Root ST produced higher microhardness values than MTA Fillapex at all root levels. In the evaluation according to root levels, the highest mean microhardness values for both materials were obtained in the middle third, whereas the lowest values were observed in the apical region. The obtained results indicate that calcium silicate-based root canal sealers may exert mechanically favorable effects on dentin tissue and that material composition may play a determining role in dentin microhardness.</p>