To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p < 0.05). Fatty acid compositions of dual specie biofilm increased in both 1% and 2% QAS specimens (p < 0.05). Quaternary ammonium silane demonstrated to be a potent antibacterial cavity disinfectant and a plaque inhibitor and can be of potential significance in eliminating caries-forming bacteria.