Aim To evaluate the nanohardness reduction and erosion in root canal dentine after application of 17% ethylenediaminetetraacetic acid (EDTA), 9% etidronic acid (HEBP) and 2% peracetic acid (PAA) either alone or combined with sodium hypochlorite (NaOCl). Methodology One hundred and sixty single-rooted teeth were decoronated and their root canals were chemomechanically prepared. For the nanohardness evaluation, 80 roots with instrumented canals were split longitudinally into two halves. Dentine blocks with a length of 7 mm were obtained from the middle third of one half of each tooth and were embedded in epoxy resin blocks exposing the root canal walls. Initial nanohardness values of root canal dentine were measured for each specimen using a nanoindenter with a Berkovich tip. The specimens were divided into seven experimental (n = 10) and one control (n = 10) group according to the final irrigation regimens: Group 1: 2.5% NaOCl, Group 2: 17% EDTA, Group 3: 2.5% NaOCl-17% EDTA, Group 4: 2% PAA, Group 5: 2.5% NaOCl-2% PAA, Group 6: 9% HEBP, Group 7: 2.5% NaOCl + 9% HEBP and Group 8: Distilled water (control). The measurement of the nanohardness values was repeated for each specimen after being exposed to the irrigants. The nanaohardness reduction of each sample was calculated and recorded. The other 80 instrumented root canals were used for scanning electron microscope (SEM) evaluation. First, they were irrigated with the same protocols stated above and longitudinally split into two halves. One half of each root canal was viewed using SEM for the amount of erosion. The erosion scores were recorded. Data were analysed statistically using Kruskal-Wallis and Bonferroni-adjusted Mann-WhitneyU-tests with a significance level of 0.05. Results Use of etidronic acid and NaOCl + HEBP reduced the nanohardness values significantly more than the other experimental solutions (P < 0.05). There was no significant difference between the values for the nanohardness reduction in the samples irrigated with a single chelator and chelator combined with NaOCl (P > 0.05). Peritubular and intertubular erosion were observed in the samples irrigated with HEBP and NaOCl + HEBP. Conclusion Final irrigation of root canals using etidronic acid alone or combined with NaOCl was associated with structural alterations in root canal dentine of extracted teeth.