Introduction: This study aimed to evaluate the effect of ultrasonic activation (UA) of endodontic sealers on dentin tubule penetration and the bond strength to root dentin. Method: One hundred single-rooted teeth were prepared with 40.06 nickel-titanium instruments and divided into 2 groups: with or without UA. Three resin-based sealers (MTA Fillapex [Angelus Dental Solutions, Londrina, PR, Brazil], Sealer Plus [MK Life Medical and Dental Products, Porto Alegre, RS, Brazil], and AH Plus [Dentsply, DeTrey GmbH, Konstanz, Germany]; n 5 20) and 2 calcium silicate–based sealers (Sealer Plus BC [MK Life Medical and Dental Products] and EndoSequence BC [Brasseler, Savannah, GA], n 5 20) were used and subdivided (n 5 10) according to the protocols. Fluo-3 (Thermo Fisher Scientific, Waltham, MA) and rhodamine B dyes were added to the calcium silicate- and resin-based sealers, respectively. In the UA groups, the activation was performed for 40 seconds followed by lateral compaction. Samples were transversely sectioned to evaluate the dentin tubule penetration and the bond strength to root dentin. The penetration data were analyzed with the Student t test, 1-factor analysis of variance, and Bonferroni tests. Bond strength was eval- uated using the Student t test, Kruskal-Wallis, and Dunn post hoc test. Results: Resin-based sealers showed the highest tubule penetration without UA (P , .05). UA significantly enhanced MTA Fillapex and Endosequence BC dentin tubule penetration (P , .05). AH Plus and Sealer Plus BC improved their bond strength to root dentin after UA (P , .05). AH Plus/ UA, Sealer Plus/UA, and Sealer Plus BC/UA presented the highest bond strength values (P , .05). Adhesive failures were predominant in all groups regardless of the use of ultrasound. Conclusions: UA interferes with tubule penetration and the bond strength to root dentin of resin- and calcium silicate–based sealers