Aim and Objective The present in vitro study was done to comparatively evaluate the effect of different endodontic irrigation protocols on the microhardness at coronal, middle, and apical levels of root canal dentin. Method The total sample size for the study was 100. Each sample consisted of a longitudinally sectioned half of a root of a single-rooted tooth, which was then embedded in acrylic resin. The prepared samples were divided randomly into five groups of twenty samples each. Each group was treated with its respective irrigant to be tested. Group I was the control—the specimens were treated with distilled water. The specimens in group II were treated with sodium hypochlorite (NaOCl), followed by ethylenediamine tetra-acetic acid (EDTA). Specimens in group III were treated with NaOCl, followed by CHX. Specimens in group IV were treated with NaOCl, followed by hydroxyethylidene bisphosphonate (HEBP), and specimens in group V were treated with NaOCl, followed by propolis. Following this, all the samples were placed on the Vickers microhardness tester, and the results were tabulated and statistically analyzed to determine the irrigant solutions' effect on the microhardness of root dentin at coronal, middle, and apical third. Results Intergroup comparison of various irrigants showed that 18% HEBP showed the least reduction in the microhardness values of root canal dentin, and 17% EDTA caused the maximum decrease in microhardness values with a significant difference. When the baseline values were compared with the values after the use of experimental solutions in all the groups, it was observed that the microhardness gradually decreased from coronal to apical third of root dentin. Conclusion On the basis of the results obtained, it can be concluded that weak chelators such as 18% HEBP do not affect the microhardness considerably, and that they can be used in future in place of strong chelators, that is, EDTA, which make root dentin weak by more microhardness reduction.