Objective: This study aimed to assess the efficacy of 0.2% Chitosan and 17% EDTA, with or without ultrasonic activation, in removing the smear layer (SL). Methods: Fifty bovine incisors were sectioned 17 mm from their apices, instrumented using ProTaper Universal instruments and divided into five groups (n=10): I- Distilled water + conventional irrigation (CI) (control), II- 17% EDTA + CI, III- 0.2% Chitosan + CI, IV- 17% EDTA + passive ultrasonic irrigation (PUI) and V- 0.2% Chitosan + PUI. The roots were split longitudinally and examined under a scanning electron microscope at 500x magnification to assess SL removal. SL presence was scored by three blinded examiners using a five-point scale. Data was analyzed using Kruskal-Wallis and Mann-Whitney U-tests (p<0.05). Results: The statistical analysis revealed significant differences in SL removal among groups (p<0.05). Group IV (EDTA + PUI) exhibited significantly higher removal efficacy than other groups (p<0.05). SL removal was highest in the cervical third compared to middle and apical thirds. Conclusions: EDTA combined with PUI demonstrated superior SL removal.