Enterococcus faecalis(E. faecalis), a gram-positive facultative anaerobic bacterium, is likely to survive root canal treatment because of its extremely high alkaline tolerance, which may contribute to the refractory nature of apical periodontitis (AP). In this study, protamine was combined with calcium hydroxide to evaluate its efficacy in killingE. faecalis. First, the antibacterial activity of protamine againstE. faecaliswas investigated. Protamine reduced theE. faecalisgrowth rate at concentrations above the MIC (250 μg/mL), but was not bactericidal at any of the concentrations tested. Next, we investigated the calcium hydroxide tolerance ofE. faecalis, using a 10% 310 medium, adjusted for pH by adding a calcium hydroxide solution. The results showed thatE. faecaliscould survive and proliferate in alkaline environments up to pH 10. However, the complete killing ofE. faecaliswas observed when protamine (250 μg/mL) was added. In addition, compared with treatment with protamine and calcium hydroxide alone, membrane damage and internalization of protamine into the cytoplasm ofE. faecaliswere enhanced. Therefore, the synergistic increase in antibacterial activity may be related to the action of both antimicrobial agents on the cell membrane. In conclusion, co-treatment with protamine and calcium hydroxide seems to be very effective in sterilizingE. faecalis, and has the potential to provide a novel control method againstE. faecalisfor root canal treatment.