Dentin bonding agents with antibacterial effects may inhibit secondary caries formation and pulp inflammation by eliminating residual bacteria in and on dentin. Therefore, the antibacterial effects of Prime & Bond NT (PB), Prime & Bond NT without fluoride (PBNF), Gluma Comfort Bond (GL), ABF, Xeno CF II (XE), 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), and 0.2% chlorhexidine were tested against Streptococcus mutans, S. sobrinus, and Lactobacillus acidophilus using the agar-diffusion method with and without bovine-dentin disks (200 mum and 500 mum thickness) placed between the bacteria and the test substances. Without dentin, ABF Primer showed growth inhibition for all bacterial strains. XE inhibited S. mutans and S. sobrinus, and PB S. sobrinus. ABF Bonding inhibited L. acidophilus. PBNF, HEMA, and TEGDMA did not have any antibacterial effects. Dentin disks of 500 mum thickness reduced the inhibitory effect of chlorhexidine to 23% to 54% compared with direct application. ABF Primer (non polymerized) produced inhibition zones against all tester strains regardless of dentin disks interposed or not. XE (against S. mutans and S. sobrinus) and PB (against S. sobrinus) did not produce any inhibition zones on 200 mum thick dentin. After polymerization, the ABF system did not inhibit bacterial growth on 200 mum thick dentin disks. A dentin barrier reduces significantly the antibacterial activity of chlorhexidine and dentin bonding agents.