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<i>Enterococcus faecalis</i> (<i>E. faecalis</i>), 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 killing <i>E. faecalis</i>. First, the antibacterial activity of protamine against <i>E. faecalis</i> was investigated. Protamine reduced the <i>E. faecalis</i> growth rate at concentrations above the MIC (250 <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">μ</mi></semantics></math></inline-formula>g/mL), but was not bactericidal at any of the concentrations tested. Next, we investigated the calcium hydroxide tolerance of <i>E. faecalis</i>, using a 10% 310 medium, adjusted for pH by adding a calcium hydroxide solution. The results showed that <i>E. faecalis</i> could survive and proliferate in alkaline environments up to pH 10. However, the complete killing of <i>E. faecalis</i> was observed when protamine (250 <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mi mathvariant="sans-serif">μ</mi></semantics></math></inline-formula>g/mL) was added. In addition, compared with treatment with protamine and calcium hydroxide alone, membrane damage and internalization of protamine into the cytoplasm of <i>E. faecalis</i> were 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 sterilizing <i>E. faecalis</i>, and has the potential to provide a novel control method against <i>E. faecalis</i> for root canal treatment.