Objectives: The present study reported a method for preparing a blend of antibacterial quaternary ammonium silanes and quaternary ammonium methacryloxy silane (QAMS) based on the sol-gel reaction between dimethyldiethoxy silane and two trialkoxysilanes, one with an antibacterial quaternary ammonium functionality and the other with a methacryloxy functionality. Methods: Reaction products of the sol-gel reaction were characterised by direct infusion mass spectrometry, FTIR and proton, carbon and silicon NMR. This blend of monomers was incorporated into an experimental universal adhesive for evaluation of antimicrobial activity against Streptococcus mutans biofilms, microtensile bond strength and cytotoxicty. Retention of quaternary ammonium species on polymerised adhesive, leaching of these species from the adhesive and the ability of resin-dentine interfaces to inhibit S. mutans biofilms were evaluated over a 3month water-ageing period. Results: The antibacterial adhesive version killed bacteria in S. mutans biofilms not only through the release of non-copolymerisable quaternary ammonium silane species (release-killing), but also via immobilised quaternary ammonium methacryloxy silane that are copolymerised with adhesive resin comonomers (contact-killing). Contact-killing was retained after water-ageing. The QAMS-containing universal adhesive has similar tensile bond strength as the control and two commercially available universal adhesives, when it was used for bonding to dentine in the etch-and-rinse mode and self-etching mode. Incorporation of the antimicrobial quaternary ammonium species blend did not adversely affect the cytotoxicity of the universal adhesive formulation. Conclusions: Instead of using quaternary ammonium dimethacrylates and nanosilver, an alternative bimodal antimicrobial strategy for formulating antimicrobial universal dentine adhesives is achieved using the one-pot sol-gel synthesis scheme. Clinical Significance: The QAMS containing universal dentine adhesives with dual antimicrobial activity is a promising material aimed at preventing second caries and prolonging the longevity of resin composite restorations. (C) 2014 Elsevier Ltd. All rights reserved.