Background and aim: Despite scientific evidence that even some microorganisms may be useful, endodontic intervention has persisted to prioritize the removal of all microorganisms from the root canal system. Indeed, information regarding the significant role of probiotic microorganisms in endodontic treatment has been sparse. This study aimed to carry out an in vitro evaluation of the antimicrobial activity of probiotics against endodontic pathogens. Methods: The evaluation was carried out in three stages. In Stage 1, the agar cup well procedure was used to analyse the efficiency of probiotics microorganisms against Enterococcus faecalis bacteria and Candida albicans microorganisms in the planktonic stage. In Stage 2, a deferred antagonistic experiment was used to determine the activity of probiotic microorganisms against endodontic pathogens like E. faecalis and C. albicans in the planktonic phase. In Stage 3, biofilm phase evaluation of an intracanal probiotic microorganism carrier was done. The region of maximum inhibition was measured at the end of Stages 1 and 2. The antimicrobial activity was recognized when the dimension of the region of maximum inhibition was 10 mm or above. The colony-forming unit/millilitre was measured at the end of Stage 3.Results: There was marked antimicrobial activity of probiotic microorganisms against the pathogenic microorganisms E. faecalis as well as C. albicans in Stages 1 and 3, i.e., during the evaluation involving agar cup and evaluation at the biofilm stage. However, no antimicrobial activity of probiotic microorganisms was observed against pathogenic endodontic microorganisms in Stage 2, i.e., during evaluation involving the use of the deferred antagonistic technique.Conclusion: It can be concluded that probiotic therapy is a promising antibacterial treatment approach that should be further investigated. This study shows that probiotics can help effectively in endodontic treatment and that more in vitro as well as in vivo research is needed to fully appreciate the advantages of bacteriotherapy in the field of endodontics.