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The purpose of this study was to evaluate the effect of acrylated hydroxyazobenzene (AHA) copolymers in a composite-resin matrix on Streptococcus mutans (SM) biofilms. Methods: The AHA was synthesized and polymerized within a bisphenol A-glycidyl methacrylate and triethylene glycol dimethacrylate (bisGMA:TEGDMA) matrix while bisGMA:TEGDMA discs served as controls. The cytotoxicity of AHA was determined using a cell viability assay. Sucrose-dependent SM biofilms were grown on the AHA and control substrates. At 24 hours and after mechanical toothbrushing (equivalent to six months), the number of live SM was quantified on the substrates and in the surrounding media. Microscopic images of the substrates were captured after live-dead staining. Results: The AHA substrates were as biocompatible as bisGMA: TEGDMA substrates. The microscopic images and quantification demonstrated no live SM on the AHA substrates and in the surrounding media as compared to the controls. The inhibitory efficacy of AHA substrates on SM biofilm was intact even after mechanical toothbrushing. Conclusions: Acrylated hydroxyazobenzene in a composite-resin matrix completely inhibits SM proliferation growth and demonstrates a zone of SM inhibition. The antibacterial propertyof AHA could be harnessed for caries prevention in high caries-risk children by incorporating AHA into the restorative and sealant materials. [ABSTRACT FROM AUTHOR]