Root canal fillings are intended to prevent microbial proliferation over time in the canal after treatment. The objective of this study was to assess biofilm proliferation within the sealer-dentin interfaces of 2 methacrylate resin-based systems, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP), aged for up to 6 months. Standardized specimens (n = 45) comprising the coronal 5 mm of human roots were filled with the test materials and gutta-percha. Specimens were either not preincubated (control, n = 9) or were incubated in sterile saline for 1 week, 1 month, 3 months, or 6 months (n = 3/group). Monospecies biofilms of Enterococcus faecalis were grown on the specimens for 7 days in a chemostat-based biofilm fermentor mimicking pathogenic oral conditions. The extent of E. faecalis proliferation within the sealer-dentin interface for each material and incubation period group was assessed by using fluorescence microscopy of dihydroethidium-stained specimens. TE had less biofilm proliferation than both EP and SE (P < .01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (P < .05). Maximum depth of biofilm penetration was recorded for SE at 1 month (P < .05). Within the test model used, the SE and EP sealers were more susceptible to interfacial biofilm proliferation than the TE restorative material. This susceptibility diminished after aging the materials' interfaces for 6 months.