This study aimed to evaluate the effectiveness of two sodium hypochlorite concentrations at different exposure times and temperatures against Enterococcus faecalis biofilms of varying ages in human dentinal tubules. Dentin blocks were infected with E. faecalis for either 3 days or 3 weeks of incubation. Subsequently, the samples were exposed to sterile water, 2%, and 5.25% sodium hypochlorite for 3 and 10 min at 20 °C and 60 °C . Viability staining and confocal laser scanning microscopy were used to assess the proportion of killed bacteria in the dentinal tubules after exposure. There are no significant differences in the efficacy of E. faecalis killing between 2% sodium hypochlorite at 60 °C for various exposure times and 5.25% sodium hypochlorite at different temperatures or exposure times (P > 0.05). When both solutions were compared at the same temperatures with a 10-min exposure time, no significant differences in the effectiveness of E. faecalis killing between 2% and 5.25% sodium hypochlorite were observed (P > 0.05). To optimize the effectiveness of sodium hypochlorite in killing E. faecalis while minimizing potential damage to root dentin and soft tissue, clinicians should prioritize increasing the temperature or exposure time of sodium hypochlorite, rather than raising its concentration.