ABSTRACT The successful of endodontic treatment affected by an effective disinfectant. Chelating agent such as ethylenediaminetetraacetic acid (EDTA), EDTAC and EDTA combined with urea peroxide were an effective disinfectant to remove smear layer. The propose of this study was to evaluate the effect of chelating agents 15% EDTA, 15% EDTAC and 15% EDTA combined with urea peroxide on the microhardness of coronal 1/3, middle 1/3 and apical 1/3 segments of root canal dentin. METHOD Thirty single rooted premolars were sectioned horizontally at cervical level. After root canal treated the roots were sectioned into three segments which then embedded in acrylic resin. Ninety sectioned root segments were devided into 3 groups of 30 Each group was treated with 15% EDTA, 15% EDTAC and 15% EDTA combined with urea peroxide respectively for 5 minutes. The groups were then devided into 3 subgroups which were coronal 1/3 segments, middle 1/3 segments and apical 1/3 segments. Root canal dentin microhardness measurement was carried out using Vickers Microhardness Tester before and after application of chelating agent. The data were statistically analized using Kruskall Wallis and Mann-Whitney U test at 95% confidence level. The results showed that there were reduction on root canal dentin microhardness after treated with chelating agents where 15% EDTA showed minimal microhardness reduction. Keywords : 15% EDTA, 15% EDTAC, 15% EDTA combined with urea peroxide, dentin microhardness, segment of root canal dentin