Aim: The aim of the present study was to evaluate and compare the antimicrobial efficacy of placentrex gel against Enterococcus faecalis (E. faecalis) in human root dentin by comparing it with two commonly accepted intracanal medicaments, that is, calcium hydroxide and 2% chlorhexidine (CHX) gluconate gel. Materials and methods: A total of 120 human-extracted, anterior, single-rooted teeth with patent root canals and fully developed root apices were used. The dentin blocks were arranged into four groups. Group I served as the control and was treated with saline. Groups II, III, and IV were tested with specific intracanal medicaments, which were placentrex gel, calcium hydroxide, and 2% CHX gel. Specimens were contaminated with a strain of E. faecalis. Antimicrobial assessment of the contaminated specimens was carried out on the 1st, 3rd, and 5th day. The collected data were analyzed using the analysis of variance (ANOVA) test followed by Tukey's post hoc test. Results: On day 1, 2% CHX gel (19%) demonstrated the strongest antimicrobial effect against E. faecalis, followed by calcium hydroxide (13%) and placentrex gel (9%). This trend persisted on days 3 and 5, with 2% CHX (day 3—16% and day 5—25%) consistently exhibiting the highest antimicrobial efficacy, followed by placentrex (day 3—9% and day 5—16%) and calcium hydroxide (day 3—6% and day 5—12%) in decreasing order. Conclusion: In the current study, 2% CHX demonstrated the highest antimicrobial efficacy, followed by placentrex, while the effectiveness of calcium hydroxide declined from the 3rd day onward. Clinical significance: The present study is clinically relevant as it addresses the critical challenge of countering E. faecalis, a key pathogen in persistent root canal infections. The results have the potential to guide clinicians in selecting optimal therapeutic agents, thereby improving the success rates of root canal treatments and enhancing overall patient outcomes in endodontic practice.