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The purpose of this systematic review was to summarize the outcomes of in vitro studies of root canal sealers for their antimicrobial effectiveness against Enterococcus faecalis on the basis of direct contact test.An exhaustive literature search was performed by using MEDLINE, Scopus, TRIP, and OpenThesis databases, followed by an extensive hand search of references of identified articles. Two independent reviewers evaluated the studies for eligibility against inclusion and exclusion criteria and performed data abstraction and risk of bias evaluations.A total of 31 articles were assessed for eligibility. Of these, 24 met the inclusion criteria and were included in the systematic review. A considerable heterogeneity was found in the methodologies of included studies. Therefore, it was not feasible to perform meta-analysis. Majority of the studies reported that different categories of freshly prepared sealers possessed some form of positive antimicrobial activity against E. faecalis for up to 24 hours. Antimicrobial efficacy was lost as the material set. Moderate evidence was found for no antimicrobial activity of aged (2-day to 7-day set) sealer samples across all categories.The evidence indicated positive antimicrobial activity of freshly mixed sealers against E. faecalis. Antimicrobial efficacy was lost as the material set, with no bacterial growth inhibition by 2-day to 7-day set sealer samples. However, the studies included in this review presented medium to high risk of bias. This review identified the need for the development of standardized methods to evaluate antimicrobial activity of root canal sealers in in vitro studies.Different sealers demonstrated different antimicrobial activity against E. faecalis.Positive antimicrobial activity of freshly mixed sealers against E. faecalis up to 24 h.Antimicrobial activity was lost as the material set with no inhibitory effect for 2–7 d.Standardizing in vitro tests for antimicrobial activity of endodontic sealers is needed.More studies are needed to evaluate sealers' effect against multiple endodontic pathogens.