Background: The smear layer acts as a physical barrier against penetration of root canal medicaments and sealers, thus compromising the seal leading to microleakage. Objectives: This study was conducted to evaluate the efficacy of 17% ethylenediaminetetraacetic acid (EDTA), 0.2% chitosan solution, and 0.2% carboxymethyl chitosan (CMC) used as smear layer removing agents in retrograde root canal preparation using scanning electron microscopy (SEM). Materials and Methodology: Eighty single-rooted teeth extracted for periodontal reasons were collected for the study. Root canals were prepared and obturated with gutta-percha coated with AH plus resin sealer. Apical 3 mm of each root resected and Class I retrograde preparation carried out using ultrasonic handpiece and ultrasonic retro tips to a depth of 3 mm along the root long axis. In Group 1 (control), normal saline solution alone was used for smear layer removal. In Group II, 17% EDTA, Group III and IV were treated with 5 ml of 0.2% chitosan and 0.2% carboxyl methyl chitosan, respectively, for 3 min. Blinded evaluation of specimens using SEM was performed independently by two operators who registered the amount of the smear layer present on the surface of the canal walls based on the score described by Hülsmann et al . Results: Group I (saline) was least efficient in the removal of the smear layer. Group II (17% EDTA), Group III (0.2% Chitosan), and Group IV (0.2% CMC) efficiently removed the smear layer from the retrograde cavity with mean scores 1.35, 1.60, and 1.35, respectively. Statistically, no significant difference found in Group II (17% EDTA), Group III (0.2% Chitosan), and Group IV (0.2% CMC). Conclusions: About 0.2% CMC and 0.2% chitosan can be better alternatives to 17% EDTA for smear layer removal due to their biological advantages.