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(1) The objective of the study is to conduct a comprehensive systematic review of in vitro studies in order to assess the depth to which E. faecalis bacteria penetrate human dentinal tubules after the use of various irrigation solutions. (2) Methods: A literature search of the MEDLINE, Scopus, Cochrane CENTRAL, and Embase databases was conducted, as well as a backward and forward citation search. Two independent reviewers then selected suitable studies based on inclusion and exclusion criteria. Data were extracted and the risk of bias and methodology of the studies were evaluated. (3) Results: Out of a total of 504 papers evaluated following the removal of duplicates, 7 studies met the inclusion criteria and were included in the systematic review. The heterogeneity of the studies made it impossible to perform a meta-analysis. The majority of the studies reported that sodium hypochlorite (NaOCl) and chlorhexidine digluconate (CHX) can affect the penetration depth of E. faecalis suspensions. The studies included in this review possess a moderate to high risk of bias and thus represent moderate evidence that the antimicrobial activity of NaOCl and CHX affects the intra-tubular penetration of bacteria. (4) Conclusions: The evidence indicates that irrigants may affect the bacteria inside human dentinal tubules. Standardized high-quality methods are needed to evaluate bacterial penetration in in vitro studies.