AbstractThe aim of this study was to determine the in vitro antibacterial effect of two experimental glass ionomer cements (GICs) on Streptococcus milleri, Staphylococcus aureus, and Enterococcus faecalis after 24 and 48 h incubation by using the agar diffusion inhibitory test. Silver zeolite (SZ) was added at 0.2 and 2% mass fraction concentration to GIC (Endion). The control group was Endion with no SZ. Each of them were prepared to uniform size using a custom‐made Teflon mold, and the GIC materials were prepared to form disks (n = 5 per group). The effect of these materials on the growth of three bacteria associated with endodontic infections was determined using the agar diffusion inhibitory test. The amounts of silver ion release from these materials were measured with atomic absorption spectrophotometry at 10 min, 24‐ and 48‐h periods. The pH of samples was measured with a pH‐meter at 10 min, 24‐ and 48‐h periods. After the incubation period, the agar plates were evaluated and the degrees of bacterial inhibition were measured in millimeters. A comparison of the mean of the test materials was statistically different in each group of specimens (p < 0.05). Between the two tested materials 2% SZ containing GIC showed the largest zone of inhibition on the agar plates of all the tested strains (p < 0.05). The most inhibition in bacterial growth occurred in E. faecalis. Adding 2% SZ to GIC resulted in a significant increase in the silver release into deionized water. This study demonstrated that GIC had an inhibitory affect on Streptococcus milleri, Staphylococcus aureus, and Enterococcus faecalis and that adding SZ increases that affect proportional to its concentration. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009