Aim To evaluate and compare the sealing ability of bio ceramic root canal sealer after ultrasonic activation of root canal irrigation solutions 3% NaOCl , 40% Citric acid & Neem leaf extract . Materials and Methods In this study, 42 mandibular premolar teeth with single roots and canals were divided into three groups for irrigation: 3% NaOCl, 40% citric acid, and Neem leaf extract. After access preparation and glide path creation with a #10 K-file (0.02 taper), the canals were instrumented using a crown-down technique to an ISO size of 30, with saline as the irrigant. Ultrasonically activated irrigation was used, followed by saline in all groups. The canals were obturated with Dentsply Gutta- percha and Bio C sealer, 1mm short of the working length, and sealed with temporary restorations. Teeth were incubated at 37°C for 3 days, and horizontal sections at 3mm from the apex were taken. Confocal laser scanning microscopy measured sealer penetration, and statistical analysis was conducted to compare results between groups. Results The results suggest that 40% citric acid is the most effective irrigant for enhancing sealer penetration into dentinal tubules at the apical third, both in terms of percentage and depth of penetration, compared to 3% NaOCl and neem leaf extract. This indicates that citric acid may improve the sealing ability of root canal treatments more effectively than the other tested irrigants. Conclusion The results suggest that 40% citric acid is the most effective irrigant for enhancing sealer penetration into dentinal tubules at the apical third, both in terms of percentage and depth of penetration, compared to 3% NaOCl and neem leaf extract. This indicates that citric acid may improve the sealing ability of root canal treatments more effectively than the other tested irrigants.