The present study aimed to evaluate the influence of the following irrigating solutions on the microhardness of root canal dentin: 2% sodium hypochlorite (2NaOCl), 5% sodium hypochlorite (5NaOCl), super-oxidized water (400 ppm Sterilox - Sx) and 17% EDTA (E). Eighty roots from bovine incisors were randomly divided into 8 groups (n=10): 2NaOCl, 5NaOCl, Sx, and 2NaOCl + E, 5NaOCl + E, Sx + E (associated with E as final irrigant for 5 min), E solely and distilled water (dH2O) as the negative control. Root canal preparation was performed by hand instruments, using one of the irrigation protocols for 30 min. Then, 5 mm of the cervical root third were cut out from each sample and subjected to the Vickers microhardness test, at two points, one at approximately 500-1000 µm from the root canal lumen (distance 1), and the other at approximately 500-1000 µm from the external root surface (distance 2). Data were analyzed by Wilcoxon and Kruskal-Wallis tests at 5% significance level. Microhardness values at distance 1 were significantly lower than those at distance 2 for all groups, except 5NaOCl and 5NaOCl + E groups (p>0.05). EDTA showed the lowest microhardness values. However, no statistically significant difference was detected among groups at distance 1 and EDTA was significantly different only from Sx at distance 2. In conclusion, all tested solutions showed lower microhardness at the most superficial root canal dentin layer compared to the one found near the external root surface, except 5NaOCl and 5NaOCl + E; EDTA promoted lower microhardness values in comparison to Sterilox at this site.