Copper nanoparticles (NCu) were synthetized and added to commercial glass ionomer cement, to evaluate in vitro its antibacterial activity against oral cavity strains. The NCu were synthesized by copper acetate reduction with L-ascorbic acid and characterized by FTIR, Raman, XPS, XRD and TEM. Then, commercial glass ionomer cement (GIC) was modified (MGIC) with various concentrations of NCu and physicochemically characterized. Cell viability was tested against human dental pulp fibroblasts (HDPFs) by Alamar-Blue assay and antibacterial test was performed against S. mutans and S. sanguinis by colony forming unit (CFU) growth method. Synthesized NCu rendered a mixture of both metallic copper and cuprous oxide (Cu2O). HDPF viability reduces with exposure time to the extracts (68–72% viability) and MGIC with 2–4 wt% NCu showed antimicrobial activity against the two tested strains.