Use of non-thermal atmospheric plasma (NTAP) brush on immobilization of dimethylaminohexadecyl methacrylate (DMAHDM) onto dentin bonding substrate, and resulting antibacterial activity against Streptococcus mutans were investigated. A bonding substrate with several-micron-demineralized layer was created from human dentin. DMAHDM was applied onto the demineralized layer with or without plasma exposure. Scanning electron microscopy (SEM) and Fourier transformed infrared (FTIR) spectroscopy were employed to verify immobilization/grafting of DMAHDM onto the substrate. Antibacterial activity of the resulting substrate was assessed by using colony-forming unit (CFU) and confocal scanning laser microscopy. Effects of saliva pellicle treatment and aging process on the above substrate were also evaluated. The SEM/FTIR results demonstrated that NTAP could induce DMAHDM immobilization onto dentin substrate, which was further verified via quantitative FTIR analysis. Comparing with non-plasma-treated, the plasmatreated substrate, with CFU 4 log lower, exhibited much stronger inhibitory effects, which were minimally affected by saliva or aging. The DMAHDM-immobilized dentin substrate showed effective and sustained antibacterial characteristics.