Introduction: This study aimed to evaluate the effects of root canal irrigants on the microhardness of root canal dentin in the presence and absence of surface-modifying agents. Methods: Forty-eight root halves were prepared by longitudinal splitting of the distal roots of 24 freshly extracted mandibular human third molars and embedded in autopolymerizing acrylic resin, leaving the dentin surface exposed. After polishing, the microhardness values of the untreated dentin surfaces were recorded by using Vickers tester at the mid-root level. The root halves were randomly assigned to 6 groups composed of 8 samples each and treated for 5 minutes with one of the following irrigants: 17% EDTA, REDTA, 2% chlorhexidine gluconate (CHX), 2% CHX with surface modifiers (CHX-Plus), 6% NaOCl, or 6% NaOCl with surface modifiers (Chlor-XTRA). After surface treatment, dentin microhardness values were recorded at close proximity to the initial indentation areas. Experimental data were statistically analyzed by using the t test and one-way analysis of variance, followed by Tukey honestly significant difference test at alpha = 0.05. Results: EDTA, REDTA, NaOCl, and Chlor-XTRA significantly decreased the microhardness of root dentin compared with intact controls (P < .05). Conclusions: The addition of surface modifiers to the irrigants did not affect the microhardness of the samples.