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This study was conducted to evaluate the efficacy of 0.2% Chitosan Nanoparticles (CNPs), CNPs/EDTA (1:1) and 17% EDTA on smear layer removal ability.Methods: Fifteen extracted human permanent mandibular premolars with single canals were decoronated and biomechanically prepared using protaper next rotary system and 2.5% Sodium hypochlorite, then randomly distributed according to the final irrigation protocol into 3 group; Group I (0.2% CNPs), Group II (CNPs/EDTA) (1:1), and Group III (17% EDTA). A standardized volume of 5 ml of each tested irrigant (s) was used for 3 minutes. Teeth were longitudinally split to evaluate the smear layer eradication using scanning electron microscope at magnifications of 500x and 1000x. The micrographs for each third were given scores by two blinded observers using Takeda., et al. (1999) scoring system.Results: There was no statistically significant difference in the scores of the three groups at the coronal, middle, and apical thirds (Pvalue = 0.368, 0.054, and 0.0900) respectively. As for the combined total scores of each group, Group I (CNPs) showing a significantly higher smear layer elimination than both Group II (CNPs/EDTA) and Group III (EDTA) (P-value = 0.044).Conclusions: None of the used chelating agents resulted in complete removal of the smear layer. The combined use of CNPs/EDTA (1:1) did not show superior performance over the other two groups.