Introduction: Penetration of endodontic sealers into dentinal tubules is essential for ensuring successful root canal treatment by creating a hermetic seal that prevents reinfection. Traditionally, 17% EDTA has been used as the final irrigant for smear layer removal. However, concerns regarding its limited antimicrobial properties and potential for dentin erosion have prompted the exploration of alternative solutions. This in vitro study compared the effectiveness of Tulsi extract with that of 17% EDTA in promoting sealer penetration within the dentinal tubules. Methodology: Thirty extracted single-rooted teeth were randomly divided into three groups: Group 1, 17% EDTA; Group 2, Tulsi extract; and Group 3, saline control. After preparation, the root canals were irrigated with their respective soluti ons, filled with gutta-percha, and sealed with a calcium hydroxide-based sealer. Sealer penetration was analyzed using scanning electron microscopy (SEM) at the 2 mm and 5 mm levels from the root apex. Results: The results showed no significant difference in sealer penetration at 2 mm among all the groups. However, at 5 mm, both EDTA and Tulsi groups exhibited significantly greater sealer penetration than the control group (p < 0.001). No significant difference was observed between the EDTA and Tulsi groups at 5 mm (p = 0.706). Conclusion: Tulsi extract demonstrated similar efficacy to 17% EDTA in facilitating sealer penetration, suggesting its potential as a biocompatible and antimicrobial alternative for root canal irrigation protocols. Further studies are warranted to explore its clinical applications and commercial viability.