The sealing ability of gutta-percha/sealer root canal filling was compared to a new thermoplastic synthetic polymer-based obturation material (Resilon), using a microleakage model and a new sequence detection assay One Cut Event AmplificatioN (OCEAN). Eighty-eight extracted human teeth, shaped with K-Files and the ProTaper Technique, were randomly assigned to four groups (n = 22) and obturated in the apical 5 mm. Group R were obturated with the Resilon/Epiphany technique; group GP were obturated with gutta-percha and Zinc oxide eugenoe sealer; group RCH and GPCH received calcium hydroxide intracanal medication before being obturated. Sterilized specimens were inoculated with Enterococcus faecalis and incubated in sterile medium for 47 days. DNA extracted from the specimens was amplified by PCR and then identified by the OCEAN technique. Samples obturated with Resilon root canal filling material showed a greater number of microleakage events than the other groups (p = 0.036). Calcium hydroxide medication did not have a relevant impact on the quality of the apical seal (p = 0.044).