INTRODUCTION: Different irrigations solutions may cause alteration in the physicochemical properties of dentin structure thereby affecting the microhardness of root canal dentin. OBJECTIVES: to evaluate the effect of different irrigation protocols on microhardness of human root canal dentin. MATERIALS AND METHODS: Forty extracted single rooted lower premolars were used. All teeth were instrumented using manual stainless steel files and irrigated by 2ml distilled water between each file, then were sectioned by longitudinal splitting of each tooth. The root halves were randomly assigned into 4 parallel groups (n=10) and immersed for 5 minutes with one of the following irrigants: Group I: 10 ml of 2.5% Sodium Hypochlorite (NaOCL), Group II: 10 ml of 17% ethylene diamine tetra-acetic acid (EDTA) followed by 10 ml of 2.5% NaOCL, Group III: 10 ml of 2.5% NaOCL followed by 10 ml of 2% chlorhexidine digluconate (CHX), Group IV: 10 ml of 2.5% NaOCL followed by 10 ml distilled water then were followed by 10ml of 2% CHX. Ten root halves from each group were prepared to measure dentin microhardness at baseline measurement and after treatment to determine the change in microhardness, using Vickers tester. RESULTS: Data were analysed using t-test, ANOVA test and Post Hoc test.Group II showed the highest percentage decrease in microhardness values, followed by group III, then group IV and the lowest was group I. All groups showed a significant difference between each other (P < 0.05), except group III and IV. The coronal third showed the highest percentage decrease with significant difference between apical and middle thirds (P < 0.05), in which there was no significant difference between them. CONCLUSIONS: CHX is the best final irrigant if there is excellent intermediate flush for prevention of its precipitation with NaOCL.The coronal third needs conservative approach as it is the most affected third.