The purpose of this study was to compare the sealing ability of MTA Fillapex, Apatite Root Canal Sealer and AH26 sealers. The present in vitro study was carried out on 142 extracted single-rooted human mature teeth. The teeth were randomly divided into three experimental groups (n=44) and two control groups (n=5). Three root canal sealers were MTA Fillapex, Apatite Root Canal Sealer and AH26. The teeth in the control groups were either filled with no sealer or made completely impermeable. The root canals were prepared and obturated with gutta-percha and one of the sealers. The teeth were sterilized with ethylene oxide gas prior to the bacterial leakage assessment using Enterococcus faecalis. Leakage was evaluated every 24 hours for 90 days. Data were analyzed with descriptive statistical methods and chi-squared test. If the data were significant, a proper post hoc test was used. Statistical significance was set at P<0.05. The positive control specimens exhibited total bacterial penetration whilst the negative control specimens showed no evidence of bacterial penetration. At the end of the study, the analysis of microleakage with chi-squared test showed no significant differences between the experimental groups (P<0.05). The results of chi-squared test analyzing the pair-wise differences between the groups considering the numerical values for leakage day indicated the lowest leakage with AH26 and the highest with Apatite root sealer. According to the results of the present study, sealing ability of AH26 was significantly higher than that of MTA Fillapex and Apatite Root Canal Sealer.