Objective: The objective of this study is to compare the effectiveness of final irrigation with chitosan, ethylenediaminetetraacetic acid (EDTA), and citric acid (CA) on a resin-based sealer (AH plus sealer [Dentsply DeTrey, Konstanz, Germany]) penetration into dentinal tubules using confocal laser scanning microscopy. Materials and Methods: Seventy recently extracted human mandibular premolars were instrumented and irrigated with sodium hypochlorite (NaOCl), then divided into four groups according to the final irrigation regimen used: (1) the EDTA group: 17% EDTA + 2.5% NaOCl, (2) the CA group: 10% CA + 2.5% NaOCl, (3) the chitosan group: 0.2% chitosan + 2.5% NaOCl, and (4) the control group: 2.5% NaOCl. All teeth were obturated using the cold lateral condensation technique with gutta-percha and AH Plus sealer labeled with fluorescent dye. The apical 2 mm of specimen was discarded, and slices were obtained for apical, middle, and coronal thirds of the root with 1 mm intervals. Maximum, mean, and percentage of sealer penetration (SP) inside tubules were measured using confocal laser scanning microscopy. Results: The percentage of SP was significantly higher in chitosan, EDTA, and CA group than control group for coronal thirds (P < 0.05), whereas there was no significant difference among all groups for middle and apical thirds. Chitosan and EDTA showed increased mean values of SP depth for middle thirds (P < 0.05). In all sections, the maximum depth of SP was significantly lower in EDTA group than other groups (P < 0.05). Conclusions: Chitosan, EDTA, and CA significantly improved the percentage of SP for coronal thirds.