Using the agar diffusion method, we conducted an in vitro study to evaluate the antimicrobial activity of mineral trioxide aggregate (MTA), new endodontic cement (NEC) and Portland cement at different concentrations against five different microorganisms. A base layer was made using Muller-Hinton agar for Escherichia coli (ATCC 10538) and Candida (ATCC 10231). For Actinomyces viscosus (ATCC 15987), Enterococcus faecalis (ATCC 10541) and Streptococcus mutans (ATCC 25175) blood agar medium was used. Wells were formed by removing the agar, and the materials were placed in the well immediately after manipulation. The plates were kept at room temperature for 2 h for prediffusion, and then incubated at 37 degrees C for 72 h. The inhibition zones were then measured. The data were analyzed using ANOVA and the Tukey test to compare the differences among the three cements at different concentrations. The positive controls showed bacterial growth, while the negative controls showed no bacterial growth. All materials showed antimicrobial activity against the tested strains except for Enterococcus faecalis. NEC created larger inhibition zones than MTA and Portland cement. This difference was significant for Portland cement (P < 0.05), but not for MTA (P > 0.05). Among the examined microorganisms, the largest inhibition zone was observed for Actinomyces group (P < 0.05). The antimicrobial activity of the materials increased with time and concentration (P < 0.05). It was concluded that NEC is a potent inhibitor of microorganism growth.