Objectives: The aim of this in vitro study was to investigate the antibacterial activities of different generation dentin-bonding systems. Method and materials: The antibacterial activities of dentin-bonding systems Optibond FL Primer, Single Bond, Clearfil SE Bond Primer, and Prompt-L-Pop were evaluated against Streptococcus mutans, Streptococcus salivarius, Lactobacillus casei, and Lactobacillus acidophilus. Cervitec (1% chlorhexidine varnish) was also examined as a positive control material. Disk diffusion tests with filter paper disks (n = 10) containing a drop of each material (20 µL) were used. After incubation at 37°C for 48 hours, the growth inhibition zones were measured in millimeters. Results: Of all the materials tested, Optibond FL Primer, Clearfil SE Bond Primer, and Prompt-L-Pop showed larger growth inhibition zones than the control material for all bacterial strains. No antibacterial effect was noted for Single Bond. The results indicated that Optibond FL Primer had the strongest effect against the S mutans and L casei, and Clearfil SE Bond Primer produced the largest inhibition zone for S salivarius and L acidophilus among the test materials. Conclusion: The antibacterial effects observed for the tested different generation dentin-bonding systems may be related to the acidic nature of the materials. Keywords: antibacterial activity, dentin-bonding systems