In recent years, nanomaterials have been used in the medical-dental field as new alternative antimicrobial agents. Bismuth subsalicylate (BSS) has been used as an antimicrobial agent, but the effect of BSS in the form of nanoparticles (BSS-nano) as a potential antimicrobial agent has not been tested, in specific against bacteria responsible for periodontal disease. The aim of this study was to evaluate the antibacterial effect of BSS-nano against oral anaerobic bacteria and to assess the safety of BSS-nano by evaluating their cytotoxicity in human gingival fibroblast (HGF-1) cells. BSS-nano were synthesized by laser ablation and were previously physico-chemically characterized using in vitro assays. The antibacterial activity was measured using the tetrazolium-based XTT assay, and cytotoxicity was determined using lactate dehydrogenase (LDH) and MTS assays in HGF-1 cells. Transmission electron microscopy of HGF-1 exposed to BSS-nano was also performed. BSS-nano was shown to have a primary size of 4-22 nm and a polygonal shape. Among the tested bacterial strains, those with a greater sensitivity to BSS-nano (highest concentration of 21.7 μg ml-1) were A. actinomycetemcomitans, C. gingivalis, and P. gingivalis. BSS-nano at a concentration of 60 μg ml-1 showed low cytotoxicity (6%) in HFG-1 cells and was mainly localized intracellularly in acidic vesicles. Our results indicate that the concentration of BSS-nano used as an effective antibacterial agent does not induce cytotoxicity in mammalian cells; thus, BSS-nano can be applied as an antibacterial agent in dental materials or antiseptic solutions.