This study aimed to investigate the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. Experiments were performed in two stages. In stage 1, experiments were conducted to examine the physical properties of three types of nanoparticulates. The antibacterial properties of nanoparticulates alone and nanoparticulates mixed with zinc oxide-eugenol-based sealer were studied. In stage 2, the ability of nanoparticulates-treated dentin to prevent bacterial adherence was examined. Zinc oxide nanoparticulates, chitosan nanoparticulates, a mixture of zinc oxide and chitosan nanoparticulates, and zinc oxide nanoparticulates with multilayered coating of chitosan were tested. This study showed that the incorporation of nanoparticulates did not alter the flow characteristics of sealer but improved the direct antibacterial property and the ability to leach out antibacterial components. There was a significant reduction in the adherence of Enterococcus faecalis to nanoparticulates-treated dentin (p < 0.05). These experiments highlighted the potential advantage of nanoparticulates in root canal disinfection.