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This study evaluated the penetration of the irrigant subject to activation after performing retro‐preparation in endodontic microsurgery. Forty mandibular premolars were prepared and filled. Subsequently, 1 mm from the root apex was cut using a multi‐blade bur and the retro‐preparation was performed. In group 1, the retro‐cavity was cleaned with 2 mL of saline and then with 2 mL of 5.25% NaOCl gel mixed with 0.1% Rhodamine B. In group 2, the retro‐cavity was cleaned with 2 mL of saline, 17% EDTA gel was activated ultrasonically for 30 s and 5.25% NaOCl gel was mixed with 0.1% Rhodamine B and activated for 30 s. After 7 days, the roots were cut at 1mm from the apex and the slices were evaluated with confocal laser scanning microscopy. The samples of group 2 showed statistically better results. The activation protocol proposed resulted in enhanced irrigant penetration when compared to the traditional. [ABSTRACT FROM AUTHOR]