This systematic review aimed to compare the efficacy of herbal agents with ethylene diamine tetraacetic acid (EDTA) in removing the smear layer during root canal instrumentation. The research question in the present study was to assess: “Is there a significant difference in reducing smear layer comparing EDTA and herbal agents?” Electronic databases (PubMed, Scopus, and Web of Science) were searched from their start dates to April 2022 using strict inclusion and exclusion criteria, and reviewed following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines. Only in vitro studies comparing herbal agents with EDTA were included in the current systematic review. Two reviewers independently assessed the included articles. A total of 625 articles were obtained from an electronic database. Eighteen papers were included for review of the full text, out of which, ten papers were excluded because they did not meet the inclusion criteria. Finally, eight articles were included in the systematic review. The present systematic review considered only in vitro studies; hence, the result cannot be completely translated to strict clinical conditions. The results of the present systematic review have shown that quixabeira, morindacitrifolia, oregano extract, and neem show better smear layer removal compared to other herbal agents, whereas they showed reduced smear layer removal when compared with EDTA. Although, it was seen that most of the included studies did not report a high quality of evidence. Hence, the present systematic review concludes that herbal agents have reported to show inferior smear layer removal when compared to EDTA. Thus, as far as herbal based alternatives are concerned, there is no highest level of evidence to state its real benefit when used as a chelating root canal irrigant.