Composite resins that are used to restore hard tissues have several drawbacks including the accumulation of biofilm on teeth and restorations. Recently, quaternary ammonium poly(ethylene imine) (QA-PEI) nanoparticles were developed for additional antibacterial activity of restorative composite resins. QA-PEI nanoparticles were synthesized from cross-linked poly(ethylene imine) that was N-alkylated with octyl halide, followed by quaternary methylation with methyl iodide. QA-PEI particles that were embedded in restorative composite resin at 1% w/w resulted in the complete growth inhibition of Streptococcus mutans. Moreover, the antibacterial activity was retained for at least 3 months. The active substances on the surface of the restorative composite resin that were incorporated with QA-PEI particles were detected by X-ray photoelectron spectroscopy (XPS) and confocal microscopy measurements. The in vitro cytotoxicity tests showed a similar effect on the viability of the cell line that was tested with composites including modified and unmodified dental composite resins. In vivo toxicity studies, which were assessed on Wistar rats by the implantation of modified composite specimens, revealed no inflammation response 1 week after the implantation of restorative composite resin that was embedded with up to 2% w/w QA-PEI.