ObjectiveWe aimed to compare the apical sealing properties of three endodontic sealers, namely, C-Root SP (C-R), iRoot SP, and GuttaFlow Bioseal (GFB)in vitro.MethodsEighty-two single-rooted premolars and anterior teeth were prepared by using M3 machine with nickel-titanium file and randomly divided into six experimental groups (n=12) and two control groups (n=5). Group A1: single-cone technique (SC)+C-R; group B1: SC+iRoot SP; group C1: SC+GFB; group A2: single-cone with ultrasonic activation (SU)+C-R; group B2: SU+iRoot SP; group C2: SU +GFB; group D: positive control group, and group E: negative control group. Dye penetration length and lateral root canal filling in each group were measured by dye penetration test. A scanning electron microscope (SEM) was used to observe the interface between gutta pertscha, root canal sealer, and dentin wall. Dye penetration length was measured and analyzed by Kruskal-Wallis test, and data on lateral root canal filling were evaluated using Chi-square.ResultsThe dye penetration length in group A1 was lower than that in groups C1 and A2 (P<0 .05) but was not significantly different from the other groups (P>0.05). Lateral root canal filling was not significantly different among all groups (P>0.05). SEM showed that GFB was slightly better than C-R and iRoot SP in binding to gutta pertcha and dentin wall.ConclusionGFB, C-R, and iRoot SP demonstrate excellent apical sealing ability. Under the conditions tested in this study, SU did not yield significantly improve the apical sealing ability of the three root canal sealers.