Article HistoryBackground: In endodontic therapy, root canal irrigants are utilized to enhance mechanical debridement. However, conventional irrigants have certain drawbacks, such as being incapable of removing the smear layer, causing tissue irritation, toxicity, and unpleasant taste, especially in children. Therefore, there is a growing necessity to explore natural alternatives, such as herbal irrigants. This study sought to evaluate the antimicrobial effectiveness of German chamomile, tea tree oil (TTO), and chlorhexidine (CHX) as root canal irrigants against E. faecalis and Streptococcus mutans. Method: Fifteen test samples of German chamomile extract, tea tree oil, and chlorhexidine were taken. Indicator strains were grown in Brain Heart Infusion (BHI) agar at 37ºC for 24 hr. In preparation for the experiment, a 15 ml base layer of BHI agar was mixed with 300µl of each inoculum and placed in individual sterilized petri plates. Once the culture medium had solidified, four wells were created on each plate and subsequently filled with the designated testing materials. This methodology was employed to ensure methodological rigor and accuracy in the experimental process. In preparation for the experiment, a 15 ml base layer of BHI agar was mixed with 300µl of each inoculum and placed in individual sterilized petri plates. Once the culture medium had solidified, four wells were created on each plate and subsequently filled with the designated testing materials. This methodology was employed to ensure methodological rigor and accuracy in the experimental process. The plates were kept for 2 hours at room temperature for the diffusion of the material and then incubated at 37ºC for 48 hours. Antibacterial tests were repeated 5 times for homogeneity of the results. Results: The comparison of antimicrobial efficacy of chlorhexidine 2%, tea tree oil, and German chamomile showed a significant difference whereas the comparison between chlorhexidine 2% and tea tree oil was statistically insignificant. Conclusion: The effective reduction in the microbial count by all three test irrigants was observed to be significant. The antimicrobial efficacy of tea tree oil was found to be better than 2% chlorhexidine and German chamomile extract.