Objective: Microorganisms are directly associated with the etiology of enamel, dentin, and pulpal pathology. Due to the growing usage of one-bottle bonding materials with resin composite restorations, as well as sealing agents with amalgam restorations, it is important that they possess antibacterial properties. In the present study, the antibacterial properties of polymerized one-bottle bonding agents were tested, using the direct contact test (DCT) and the agar diffusion test (ADT). Method and materials: Quadruple samples of the following materials were tested in both assays: Bond-1, OptiBond Solo, One-Step, Gluma, Prime & Bond NT, and Synergy. In the DCT, samples were placed on the sidewalls of wells of a 96-microtiter plate and polymerized. A 10-µL suspension of Streptococcus mutans was placed on the surface of each sample for 1 hour at 37°C. Fresh media was then added, and bacterial growth was followed with a temperature-controlled spectrophotometer. In the ADT, samples were placed in punched wells of inoculated agar plates, and halos in the bacterial lawn were measured after 72 hours. Results: In the DCT, all the tested bonding agents exhibited potent antibacterial properties; virtually no viable bacteria were present in any of the samples. When the samples were aged in phosphate-buffered saline for 24 hours, all the tested agents, similar to the freshly polymerized samples, exhibited potent antibacterial properties. This property was lost in samples aged for 7 days. Fresh samples assayed by ADT demonstrated no inhibition halo around any of the samples. Conclusion: Collectively, the present data suggest that one-bottle bonding agents possess in vitro antibacterial properties for at least 24 hours. This phenomenon was demonstrated only by DCT.