Purpose:The purpose of this study was to evaluate the effect of diode laser-980nm on microhardness of young and old canal dentin after treatment with NaOCl, NaOCl/ EDTA and distilled water solutions. Material and Methods Forty-eight single rooted premolars were divided into three groups according to the chemical solutions used (16 each) (8 for young teeth <30 years old and 8 for old teeth >50 years old). Group I: irrigated with 5ml of 2.6% NaOCl activated with diode laser, group II: irrigated with 5ml of 2.6% NaOCl without laser activation followed by 5ml of 17% EDTA activated with diode laser and group III: irrigated with 5ml of distilled water activated with diode laser. Then microhardness was evaluated at coronal, middle, and apical thirds of each root canal. Results: In all young and old groups, the coronal third had the highest mean value, followed by the middle, and the apical third had the lowest mean value, with a statistically significant difference between thirds of the groups (p0.001<). There was a statistically significant difference between young and old teeth at all levels (coronal, middle, and apical) for each group (I, II, and III), with old teeth having a greater mean microhardness value than young teeth. Conclusion: Irradiation of NaOCl/EDTA solutions with 980-nm diode caused reduction in microhardness of young dentin more than old dentin, and NaOCl only or distilled water in all thirds of groups.