Purpose: Secondary caries can occur around the restoration, fixed prosthesis, and orthodontic band margins because of cariogenic bacteria. Long-lasting antibacterial effectiveness of dental cements used contemporarily can reduce this phenomenon. The purpose of this study was to examine the antibacterial activities of nine dental cements (BisCem®, Super-Bond C&B, Rely XTM, PanaviaTM F 2.0, Variolink® II, UnitekTM Multi Cure, Multilink® Automix, ClearfilTM Esthetic Cement, TransbondTM LR) using agar diffusion test. Materials and methods: The test materials were inserted into the wells of Muller Hinton agar plates inoculated with Streptococcus mutans and Streptococcus salivarious. The diameters of the inhibition zones produced around the materials were measured after 24 h of incubation. Two-Way ANOVA, the Kruskal-Wallis, and the Mann-Whitney U tests at a significance level of p < 0.05 were analyzed for the results. Results: Unitek Multi Cure cement exhibited a significant difference from the control group against both S. mutans and S. salivarious (p < 0.05). While the zone of inhibition of Unitek Multi Cure cement was shorter (8.50 ± 1.77) than the control group (12.63 ± 1.30), Unitek Multi Cure had antibacterial effect against S. mutans and S. salivarious (p < 0.05). S. mutans displayed a significantly lower resistance to Unitek Multi Cure, BisCem, and Superbond C&B than S. salivarious (p < 0.05). Conclusion: Conventional glass ionomer cement, Unitek Multi Cure, exhibited greatest in vitro antibacterial activity against both S. mutans and S. salivarious. Formation of dental caries had been suppressed by the fluoride-releasing GICs.