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Background: In this ex vivo study, the aim was to evaluate the effects of ALAD and red light on Enterococcus faecalis in infected root canals using a special intracanal fiber. Methods: A total of 70 extracted, single-rooted teeth were used. The teeth were decoronated at the length of the roots to approximately 15 mm and then instrumented. The apical foramen was sealed by composite resin, and the root canals were infected with a pure culture of E. faecalis ATCC 29212 for eight days at 37 °C. Following the contamination period, the roots were divided into seven groups, including the positive and negative control groups, and treated as follows: ALAD 45 min; red light activation 7 min; ALAD 45 min and red-light activation 7 min; sodium hypochlorite 2.5% 15 min; sodium hypochlorite 1% 15 min. The samples were taken by three sterile paper points, transferred to tubes containing 1 mL of PBS, and immediately processed for the number of colony-forming units and the cell viability by using live/dead. Results: The best treatment is obtained with 2.5% NaOCl. Except for ALAD + red light vs. 1% NaOCl, a statistically significant difference is recorded for all treatments. The combination of 2.5% NaOCl and ALAD + 7 min irradiation produces an evident killing effect on the E. faecalis cells. On the other hand, 1% NaOCl is ineffective for the viability action, with 25% of dead cells stained in red. Conclusions: This ex vivo study shows that ALAD gel with light irradiation is an efficacious protocol that exerts a potent antibacterial activity against E. faecalis in infected root canals.