The objective of this work is to prepare and verify the antimicrobial properties of tissue conditioner containing silver doped bioactive glass nanoparticles (Ag-BG NPs) in vitro against three oral pathogens. A novel bioactive glass (BG) nanoparticles (NPs) with different Ag weight percentages of 0.6, 1.2, 3 and 4 wt% was prepared via a modified sol-gel method. Characterization of Ag-BG NPs was carried out using transmission electron microscopy (TEM), Fourier transformer infrared spectrophotometer (FTIR). Antimicrobial activities of the NPs were estimated. Also, Ag + ions release was measured using an inductively coupled plasma optical emission spectroscopy (ICP/OES). The 4% Ag-BG NPs was incorporated into the tissue conditioner (TC) for the preparation of the composite specimens (TC/Ag BG 4%). The dispersion of the NPs was evaluated using SEM/EDX. Ag + ion release and antimicrobial activities were evaluated. Finally, cell cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on normal human fibroblast (BJ1). Data analysis was performed using one-way ANOVA, and Tukey's post hoc test. The Ag-BG NPs (4%) exhibited the highest statistical significant mean inhibition value against selected pathogens. A novel Ag-BG NPs with antimicrobial properties with gradual release of Ag + were developed. However, the antimicrobial activity was lost when it was incorporated into the tissue conditioner.