To evaluate bacterial leakage of Apexit Plus, a new root canal sealer, in comparison with AH Plus. A total of 56 single-rooted human teeth were randomly divided into two experimental groups of 16 roots and two control groups. Roots were filled by lateral condensation with Gutta-percha and AH Plus or with Gutta-percha and Apexit Plus. A split chamber microbial leakage model was used in which Streptococcus mutans placed in the upper chamber could reach the lower chamber only through the filled canal. Positive controls were filled only with Gutta-percha and tested with bacteria, whereas negative controls were sealed with wax to test the seal between chambers. Additionally, film thickness, solubility and dimensional change were determined. All positive controls leaked within 24 h, whereas none of the negative controls leaked after 30 days. Apexit Plus had significant less bacterial leakage (log-rank test, P < 0.0001) than AH Plus. AH Plus (0.3% solubility) showed a slightly lower solubility than Apexit Plus (0.5% solubility) but a larger film thickness (28 vs. 11 mum) according to ISO 6876:2001. Apexit Plus had a better sealing ability in comparison with AH Plus.