Aim: The aim of the study was to compare the ability of three endodontic sealers, Endofill (END), AH Plus (AHP), and Sealer Plus BC (SPB), to penetrate dentinal tubules. Materials and methods: Forty-five human teeth, single-rooted and previously instrumented mandibular premolars, were randomly divided into three experimental groups (n = 15): END (n = 15), AHP (n = 15), and SPB (n = 15). After obturation, dental sections were performed horizontally, at 2 and 5 mm from the root apex. The samples were analyzed by scanning electron microscopy associated with cathodoluminescence. Percentage penetration (PP%) and maximum penetration depth (MPD) of the sealers were evaluated by the Kruskal-Wallis and Mann-Whitney tests, for general and paired data, respectively. The Wilcoxon test was applied to analyze the differences between the 5 and 2 mm distances. A 5% significance level was adopted. Results: As for PP%, AHP and SPB were similar (p = 0.127) and presented higher values than END (AHP, p = 0.024 and SPB, p <0.001); with regard to MPD, AHP and SPB did not differ either (p = 0.450), but were higher than END (p <0.001); in both analyses, penetration was greater at 5 mm than at 2 mm (p <0.001). Conclusion: SPB showed satisfactory performance in penetrating dentinal tubules, being similar to AHP, and superior to END. Clinical significance: Greater penetration of sealer into the dentinal tubules may increase the chance of successful endodontic treatment.