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Background: One-visit apexification is a treatment of choice in necrotic immature open apex teeth. Calcium silicate base materials are suitable for this method. The purpose of this study was to investigate and compare the sealing efficiency of Biodentine, mineral trioxide aggregate (MTA) ProRoot, and calcium-enriched mixture (CEM) cement orthograde apical plug using bacterial leakage method. Materials and Methods: In this in vitro study a total of 70 extracted maxillary incisors were cleaned and shaped. A 1.1-mm standardized artificially open apex was created in all samples. The teeth were randomly divided into three experimental groups of 20, and two negative and positive control groups of 5. In experimental groups, 4-mm thick apical plugs of ProRoot MTA, CEM cement, or Biodentine were placed in an orthograde manner. Negative control samples were completely filled with MTA while positive control samples were left unfilled. Sealing efficiency was measured by bacterial leakage method, and results were analyzed by Kaplan–Meier and Chi-square tests. The level of significance was set at 0.05. Results: The highest number of turbidity was recorded for ProRoot MTA samples, while the lowest for Biodentine. There was a significant difference in the number of turbidity between ProRoot MTA and Biodentine groups (P < 0.001), but there was no significant difference between CEM cement and Biodentine (P = 0.133) and ProRoot MTA (P = 0.055). Conclusion: Within the limitation of this in vitro study, Biodentine showed promising results as a substance with good-sealing efficiency.