Objective:This study aims to evaluate and compare the antimicrobial efficacy of three selected root canal irrigants (BioPure MTAD, metronidazole, aztreonam) against microbes commonly isolated from polymicrobial microbiota of root canal infection.Materials and Methods:This study was designed with four experimental groups (Group I – Bacteroides fragilis, Group II – Propionibacterium acnes, Group III – Enterococcus faecalis, Group IV – Candida albicans) based on the microbes selected for the study. Group I and Group II bacteria were used to compare and evaluate antimicrobial effect of BioPure MTAD, metronidazole, aztreonam, and normal saline. Group III and Group IV bacteria were used to compare and evaluate antimicrobial efficacy of BioPure MTAD, aztreonam, and normal saline. Normal saline was used as a control irrigant in this study. Agar disc diffusion method was applied to assess and compare the antimicrobial action of selected irrigants.Results:Metronidazole was found to be the most effective root canal irrigant against B. fragilis and P. acnes among the tested irrigants. Mean zone of inhibition against E. faecalis has been shown to be maximum by BioPure MTAD, followed by aztreonam. Antifungal effect against C. albicans was only shown by BioPure MTAD.Conclusions:Overall, BioPure MTAD is the most effective root canal irrigant as it has shown an antibacterial effect against all the tested microorganisms. However, metronidazole showed maximum antibacterial effect against obligate anaerobes. Aztreonam also showed an antibacterial effect in the present study, raising its possibility to be used as a root canal irrigant in the future.