Wei Fan,1,* Daming Wu,1,* Franklin R Tay,2 Tengjiao Ma,1 Yujie Wu,1 Bing Fan1 1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, People’s Republic of China; 2Department of Endodontics, Georgia Regents University, Augusta, Georgia, USA *These authors contributed equally to this work Abstract: Mesoporous calcium-silicate nanoparticles (MCSNs) are advanced biomaterials for controlled drug delivery and mineralization induction. Nanosilver-incorporated MCSNs (Ag-MCSNs) were prepared in the present study using both the adsorption and template methods. Both versions of Ag-MCSNs showed characteristic morphology of mesoporous materials and exhibited sustained release of ions over time. In antibacterial testing against planktonic Enterococcus faecalis, Ag-MCSNs showed significantly better antibacterial effects when compared with MCSNs (P<0.05). The Ag-MCSNs aggregated on the dentin surface of root canal walls and infiltrated into dentinal tubules after ultrasound activation, significantly inhibiting the adherence and colonization of E. faecalis on dentin (P<0.05). Despite this, Ag-MCSNs with templated nanosilver showed much lower cytotoxicity than Ag-MCSNs with adsorbed nanosilver (P<0.05). The results of the present study indicated that nanosilver could be incorporated into MCSNs using the template method. The templated nanosilver could release silver ions and inhibit the growth and colonization of E. faecalis both in the planktonic form and as biofilms on dentin surfaces as absorbed nanosilver. Templated Ag-MCSNs may be developed into a new intracanal disinfectant for root canal disinfection due to their antibacterial ability and low cytotoxicity, and as controlled release devices for other bioactive molecules to produce multifunctional biomaterials. Keywords: antibacterial effect, mesoporosity, root canal, silver