Purpose: this study was directed to investigate the effect of 0.2% chitosan, 0.2% nano chitosan, and 5% apple vinegar compared to 17% EDTA on intraradicular dentin surface roughness. Materials and Methods: Twenty single-rooted human teeth were used in this study, after decoronation, working length determination, and mechanical preparation using ProTaper Next rotary files with irrigation after each file using 5ml of 2.6% NaOCl, the Specimens were randomly divided into four groups according to the final irrigating solution. Group I used 17%EDTA, Group II with 0.2% chitosan, Group III with 0.2% nano-chitosan, and Group IV with 5% apple vinegar. Specimens were sectioned longitudinally and evaluated for surface roughness changes using Environmental Scanning Electron Microscope (ESEM) and a photomicrograph was analyzed using a 3D software system. Results: the highest mean value of surface roughness was recorded in group IV treated with 5% apple vinegar with a statistically significant difference from the other groups (P value< 0.05). The lowest mean value was observed in group III used 0.2% nano-chitosan followed by group II irrigated with 0.2% chitosan which was lower than the control group I used 17% EDTA with a statistically significant difference between the three groups (P value< 0.05). Conclusion: 0.2% nano-chitosan as the final irrigating solution had minimal drawbacks on dentin micro-structures by producing a slight change of surface roughness.