Aim The aim of this study was to evaluate the effect of two different concentrations of chitosan irrigation with two rotary systems (Revo-S and One Shape) on smear layer removal during root canal treatment using scanning electron microscope. Materials and methods Forty freshly extracted human premolar with single canal and nearly straight roots were collected. Teeth were decoronated to obtain a standardized root length of nearly 15 ± 1 mm. Samples were randomly divided into four main groups (n = 10) according to rotary preparation system and final chitosan irrigation concentration used. Group I: Revo-S rotary system and 5 ml of 0.2% chitosan solution for 3 min as final irrigation. Group II: One Shape single file and 5 ml of 0.2% chitosan solution for 3 min as final irrigation. Group III: Revo-S rotary system and 5 ml of 0.37% chitosan solution for 3 min as final irrigation. Group IV: One Shape single file and 5 ml 0.37% of chitosan solution for 3 min as final irrigation. During preparation and after change of each succeeding file, root canal irrigated with freshly prepared 5 ml of 2.5% sodium hypochlorite solution. Then, rinsed with 5 ml of distilled water. Finally, root canals were irrigated with final chitosan irrigation concentration as mentioned in groups. Samples were split longitudinally and examined by scanning electron microscope analysis. Root canal cleanliness was qualitatively assessed at the coronal, middle, and apical regions of root halves. Kruskal–Wallis test was used to compare between final irrigation solutions at each section and Mann–Whitney U test was used for pair-wise comparisons between groups whenever indicated. The significance level was set at P value less than or equal to 0.05. Results It showed that there were no statistical significant differences between groups for smear layer covering the dentinal surfaces. When comparing different root levels, there were no statistical significant differences between coronal versus middle levels but between coronal versus apical and middle versus apical statistical significant differences were found. Conclusions Smear layer can be removed effectively in all tested groups from coronal and middle levels compared to apical level. Using multiple file system (Revo-S files) is better than single file system (One Shape file) in removal of the smear layer of root canal. Increasing the concentration of chitosan to 0.37% promoted removal of the smear layer better than 0.2% of chitosan.