The purpose of this study was to evaluate the effect of 0.2% chlorhexidine gluconate on the microhardness and roughness of root canal dentin compared with widely used irrigation solutions. Ninety, mandibular, anterior teeth extracted for periodontal reasons were used. The crowns of the teeth were removed at the CEJ. The roots were separated longitudinally into two segments, embedded in acrylic resin, and polished. A total of 180 specimens were divided into 6 groups of 30 teeth at random according to the irrigation solution used: group 1: 5.25% NaOCl for 15 min; group 2: 2.5% NaOCI for 15 min; group 3: 3% H 2O2 for 15 min; group 4: 17% EDTA for 15 min; group 5: 0.2% chlorhexidine gluconate for 15 min; and group 6: distilled water (control). Each group was then divided into 2 subgroups of 15 specimens: groups 1a, 2a, 3a, 4a, 5a, and 6a were submitted to Vickers microhardness indentation tests; groups 1b, 2b, 3b, 4b, 5b, and 6b were used for determination of the roughness of root dentin. The data were recorded as Vickers numbers and Ra, μm for roughness test. The results were analyzed statistically by using one-way ANOVA and Tukey tests. The results indicated that all the irrigation solutions except chlorhexidine significantly decreased microhardness of root canal dentin (p < 0.05); 3% H2O2 and 0.2% chlorhexidine gluconate had no effect on roughness of the root canal dentin (p > 0.05). Although there are many other factors for irrigation solution preference, according to the results of this study, 0.2% chlorhexidine gluconate seems to be an appropriate endodontic irrigation solutions because of its harmless effect on the microhardness and roughness of root canal dentin.