OBJECTIVE Confocal laser scanning microscopy (CLSM) was used to investigate the penetration of endodontic sealers into the dentinal tubules after retreatment using two different obturation techniques. METHODS Thirty mandibular premolars were prepared up to instrument F3 (ProTaper Universal, Dentsply) and filled with Endofill using the single cone technique. The canals were retreated using Mtwo instruments. Reobturation was performed with the Bio-C sealer mixed with a fluorophore dye (Fluo-3) using either the lateral condensation technique (group LC) or the single cone technique (group SC) (n = 15). Teeth were sectioned 2, 4, and 6 mm from the apex and analyzed with CLSM to assess the penetration of the sealer into the canal perimeter and the maximum depth of penetration of the sealer into the dentinal tubules. Data were analyzed using ANOVA and the Student-t and Holm-Sidak tests. RESULTS In the apical segment, the penetrated perimeter was significantly higher in the LC group than in the SC group (p < 0.05); no significant difference was found in the middle and cervical segments (p > 0.05). In terms of penetration depth, no significant differences were found for any of the segments studied (p > 0.05). CONCLUSIONS The LC technique promoted a higher percentage of canal circumference penetrated by the sealer than the SC technique in the apical segment after endodontic retreatment. CONCLUSIONS CLSM demonstrated that the LC technique promoted a higher percentage of canal perimeter penetrated by the Bio-C sealer than the SC technique in the apical segment of mandibular premolars after retreatment.