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Background The purpose of this study was to investigate the antimicrobial activities of four endodontic sealers (GuttaFlow2, AH Plus, ProRoot MTA and RealSeal) against E. feacalis , E.coli and C.albicans . Methods The antimicrobial activities of four endodontic sealers were assessed by both agar diffusion test (ADT) and direct contact test (DCT) in this study. In ADT, the results were reported as the diameter of the growth inhibition zone. Both fresh and 1-day-setting sealers were measured. In DCT, microorganisms in suspension were exposed to the sealers for 10, 30 and 60 min and the survival of microorganisms were determined after exposure at different time points(after mixing, 1 and 7 days). The number of colony-forming unit (CFU) was counted. The results were analyzed with ANOVA and Tukey tests. Results Both ADT and DCT results showed that Guttaflow2 presented no effect against any tested microorganisms. In ADT, fresh RealSeal had the largest inhibition zone against all tested microbes, followed by AH Plus and ProRoot MTA. ProRoot MTA demonstrated inhibition zones against all the three test microbes after setting for 1 day, while the other three sealers showed no inhibition activity. In DCT, fresh AH Plus had the strongest antimicrobial effects against all the three tested microorganisms for every contact times, while its antimicrobial activity diminished significantly with time. Fresh RealSeal and ProRoot MTA also showed strong antimicrobial effect and still showed antimicrobial effect after 1-day-setting. The antibacterial effects of RealSeal against E. faecalis and antifungal effect of ProRoot MTA were observed after 7 days of setting. Conclusions GuttaFlow2 had no antimicrobial activity. Freshly mixed RealSeal and AH Plus demonstrated strong antimicrobial effects. RealSeal showed antimicrobial effects after setting in DCT. ProRoot MTA showed high antimicrobial activity and exhibited anti-inflammation potentials after setting.