Background  The present study compared the effect of post space preparation time on the apical seal of two different sealers. Methods In the in vitro study, 94 central incisors were used. After the samples’ root canal preparation, they were randomly assigned to four experimental groups (n = 21). The samples in groups 1 and 2 were obturated with AH Plus sealer, gutta-percha, and in groups 3 and 4 with Endoseal MTA bioceramic sealer and single cone technique. The post spaces in groups 1 and 3 were prepared immediately and in groups 2 and 4 with a delay. The samples were evaluated at 7-, 30-, and 90-day intervals for apical microleakage using the fluid filtration technique. The data were analyzed with SPSS 25, using three-way ANOVA and independent t-test. Results  The apical microleakage in groups 3 and 4, obturated with Endoseal MTA bioceramic sealer and prepared immediately and after a delay, respectively, was not significantly different between the interval times. In group 2, obturated with AH Plus sealer and prepared for post space with a delay, the apical microleakage was significantly less than all the other groups. Group 1, obturated with AH Plus sealer and prepared for post space immediately, exhibited the least microleakage after seven days, but its microleakage increased over time to reach the level of groups 3 and 4. Conclusion  According to the results, the apical microleakage in the AH + sealer group and the delayed post-space preparation method, was significantly less than all the other groups over time.