Background: Enterococcus faecalis is the most persistent organism in the root canal which resists most of the intracanal medicaments. There is always a constant attempt to eliminate this endodontic pathogen from the root canal system. Aim: The aim of this study was to evaluate the efficacy of the association of different concentrations of proton-pump inhibitor (PPI) (Lansoprazole) with calcium hydroxide (CH) and chlorhexidine (CHX) against E. faecalis using a broth dilution method. Materials and Methods: E. faecalis was inoculated into brain–heart infusion broth at 37°C for 5 h. The master broth was then treated with CH (Group 1); CH + 2% CHX (Group 2); CH + PPI 6.25 μg/ml (Group 3A); CH + PPI 25 μg/ml (Group 3B); 2% CHX + PPI 6.25 μg/ml (Group 4A); 2% CHX + PPI 25 μg/ml (Group 4B); CH + 2% CHX + PPI 6.25 μg/ml (Group 5A), and CH + 2% CHX + PPI 25 μg/ml (Group 5B). The groups were spectrophotometrically analyzed at 630 nm at 24 h to determine the group with the least optical density. Statistical Analysis: Comparison between the groups was done by the one-way analysis of variance and Kruskal–Wallis test for multiple comparisons. Results: The mean percentage inhibition of E. faecalis by Group 5A (CH + 2% CHX + PPI 6.25 μg/ml) was the highest compared to other groups. The lowest mean value was observed in Group 3A (CH + PPI 6.25 μg/ml) indicating least efficiency. Conclusion: There was a concentration-dependent effect of PPI on CH and CHX against E. faecalis . The maximum efficacy was found when the lower concentration of PPI was associated with CH/CHX mixture.