Context: Effective endodontic treatment relies heavily on proper instrumentation, thorough disinfection, and precise three-dimensional obturation of the root canal system. Aims: This study aims to evaluate and compare continuous chelation (etidronic acid and sodium hypochlorite [NaOCl]) and sequential chelation (citric acid and NaOCl) on the dentinal tubule penetration of bioceramic-based sealer (Bio-C)-A confocal laser scanning microscopic study. Settings and Design: In vitro experimental study. Subjects and Methods: Sixty extracted permanent single-rooted teeth were selected and decoronated below cementoenamel junction to get a standardized length of 12 mm across all samples followed by enlargement of root canals till rotary ProTaper F3. The samples were randomly divided into three groups: Group I (control): Canals were irrigated with 3 mL 17% ethylenediaminetetraacetic acid/3 mL 3% NaOCl; Group II: Canals were irrigated with 10 mL etidronic acid + NaOCl solution; and Group III: Canals were irrigated with 3 mL 10% citric acid followed by 3 mL 3% NaOCl. 0.1% rhodamine B dye was mixed with Bio-C sealer followed by obturation. All the samples were subjected to confocal laser scanning microscopy evaluation. Statistical Analysis Used: One-way analysis of variance with the Bonferroni post hoc test was used for the statistical analysis. The level of significance was set at P < 0.05. Results: The pairwise comparison of depth of penetration showed statistically significant results in all the coronal, middle, and apical thirds. Group II showed better depth of penetration than Groups I and III. Conclusions: Continuous chelation protocol using etidronic acid and NaOCl showed greater and statistically significant sealer penetration depth when compared to the sequential chelation protocol, i.e., citric acid at all three levels.