Background and Objective: Enterococcus faecalis is strongly associated with persistent endodontic infections and has been reported in 24–77% of failed root canal treatments due to its ability to survive harsh environments, penetrate dentinal tubules, and form mature biofilms. Calcium hydroxide (Ca (OH)₂) is widely used as an intracanal medicament but often fails to eliminate E. faecalis. Totarol, a natural phenolic diterpene, exhibits potent antibacterial activity against Gram-positive bacteria. The aim of this study was to compare the antibacterial and antibiofilm effects of totarol and Ca(OH)₂ against clinically isolated E. faecalis using in vitro testing and an ex vivo 21-day biofilm model.

Materials and Methods: Clinical isolates were microbiologically confirmed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of totarol were determined using broth dilution and expressed in volumetric units due to formulation constraints. Agar diffusion assay was performed (n=10/group). A 21-day intraradicular biofilm model was developed in human teeth followed by 3-day intracanal medication using commercial Ca (OH)₂ paste and totarol. Dentin shavings were collected from standardized canal walls, and colony forming units (CFU) were quantified and analyzed statistically.

Results: Totarol (MBC 22.5 µl/ml) produced a significantly larger inhibition zone (38.26 ± 1.84 mm) than Ca (OH)₂ (15.69 ± 1.73 mm; p<0.0001). In the biofilm model, totarol reduced bacterial load by 97.8%, significantly greater than Ca (OH)₂ (81.1%; p<0.0001, n=10).

Conclusion: Totarol demonstrated a substantially greater antibacterial and antibiofilm effect than Ca (OH)₂ and may represent a potential adjunct intracanal medicament for persistent infections, pending further biological and clinical evaluation.