Introduction: Microorganisms induce a variety of infections and diseases in the human body and are largely ubiquitous in the nature of the contamination, directly, or indirectly leading to transmission of infectious agents. A substantial number of bacterial species have been identified as inhabitants of the oral cavity. Infection control is a major topic of concern in medical and dental healthcare settings. Aim: To evaluate the disinfecting ability of various herbal extracts on sterilization of endodontic files against endodontic pathogens. Materials and methods: The study was conducted in three phases to evaluate the disinfecting ability of tea tree, neem, eucalyptus, and ajwain oil on sterilization of endodontic files against Staphylococcus aureus, Streptococcus β-hemolyticus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Peptostreptococcus species, and Bacteroides fragilis. Results: Colony-forming units (CFU) of all tested bacteria among group I (tea tree oil) were found to be significantly lower than that among group II (ajwain oil), group III (eucalyptus oil), and group IV (neem oil), respectively. Conclusion: The antimicrobial activity among all the essential oils was found to be maximum for tea tree oil, followed in descending order by ajwain, eucalyptus, and neem oils. Hence, the incorporation of these herbs in dental practice will prove to be a valuable adjunct in dental treatment. Clinical significance: Herbs have also been long used in various endodontic medicaments and dressings with obtundent and soothing effects and exhibited a great microbial inhibition potential against the tested endodontic pathogens.