Background: Antibacterial action of root canal filling is an important factor for successful root canal treatment, so the aim of the study was to identify and to compare the antimicrobial effect of new sealer (GuttaFlow) to commonly used endodontic sealers (AH Plus, Apexit and EndoFill) against four endodontic microbes. Materials and methods: Twenty patients aged (30-40) years with infected root canals were selected. Four types of microorganisms were isolated from root canals (E faecalis, Staphylococcus aureus, E coli and Candida albicans) and cultured on Mueller Hinton agar Petri-dishes. After identification and isolation of bacterial species, agar diffusion method was used to assess the antibacterial action of four contemporary endodontic sealers used in root canal obturation (AH Plus, Apexit, EndoFill and GuttafFlow). Four wells measuring (5mm depth and 4mm diameter) were created in each Petri dish and sealer was applied into them incubated overnight at 37 C° for bacterial species and 48 hr. at 37 C° for Candida albicans prior to determination of results. Zones of inhibition (no growth of bacteria) were examined around the wells containing sealer & diameters of the zones were measured in mm. The mean of inhibition zones for each group was measured and statistically analyzed among groups using ANOVA and between groups using LSD tests. Results: There was a highly significant difference (P<0.001) among all the tested groups. EndoFill showed the maximum antibacterial action against tested microorganisms. GuttaFlow showed moderate to weak antimicrobial effect, Apexit had weak effect, while AH Plus had no antibacterial action. Conclusion: All the tested materials except AH Plus had antibacterial efficacy against E faecalis, Staphylococcus aureus, E coli and Candida albicans. EndoFill had favorable results among tested sealers and E faecalis was the most resistant bacteria, but none of the materials totally inhibited microbial growth. Thus, endodontic treatment must be performed under aseptic conditions.