The aim of this study was to evaluate the influence of different activation techniques on dentin tubule penetration of root canal sealer. Seventy‐five teeth with single canals were chemomechanically prepared. A calcium silicate‐based sealer was stained with a fluorescent dye (rhodamine B), placed into the canals and activated according to the following groups: control (no activation), EDDY, EndoActivator, ultrasonic and XP‐Endo Finisher. Then, the samples were obturated. The percentages of sealer penetration at various depth levels of root sections were measured with confocal laser scanning microscopy. XP‐Endo Finisher presented the highest penetration at 50 μm (p < 0.05). XP‐Endo Finisher showed similar penetration with EDDY at 100 and 200 μm (p > 0.05) while presented higher penetration than the other groups (p < 0.05). At 500 μm, XP‐Endo Finisher presented higher penetration than EndoActivator (p < 0.05) while similar penetration with the other groups (p > 0.05). XP‐Endo Finisher can be recommended for activation during sealer placement for better penetration into dentin tubules.