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A dentin-bonding root canal sealer (ZUT) has been developed, consisting of an experimental glass ionomer cement (KT-308) and an antimicrobial silver-containing zeolite (0.2% by weight). This in vitro study evaluated the ability of ZUT used with or without gutta-percha, to resist bacterial ingress of Enterococcus faecalis over a period of 90 days. Canals of 80 single-rooted teeth were prepared with apical patency and filled as follows (n = 10): KT-308 alone; KT-308 with a single gutta-percha cone (SCGP); ZUT alone; ZUT with SCGP; AH26 alone; AH26 with SCGP; positive control—no root canal filling; and negative control—no root canal filling, with the apices of this group sealed with C&B Metabond cement. Teeth were coated with nail polish except for the apical 2 mm, and each tooth was sealed in a 4-ml glass vial, with an 18-gauge needle inserted through the vial cover and bonded into the pulp chamber with C&B Metabond cement. After sterilization with 2.5 Mrad γ-radiation, Brain Heart Infusion broth with phenol red was injected into each vial. An inoculum of E. faecalis was pipetted through the needle into the pulp chamber every 5 days, and the broth was monitored daily for color change and turbidity. When change occurred, the broth was cultured for growth of E. faecalis. Kaplan-Meier survival analysis and the log–rank test revealed no significant differences among the three sealers used. The presence of gutta-percha, however, significantly improved resistance to bacterial ingress through obturated root canals (χ, p < 0.05). Under the conditions of this study, the hypothesized advantage of ZUT (0.2% zeolite) was not demonstrated.