In this work, we present an analysis of the antibacterial activity of TiS3nanostructures in water and 0.9% NaCl solution suspensions. TiS3nanoribbons 1–10 µm long, 100–300 nm wide, and less than 100 nm thick were produced by the direct reaction of pure titanium powder with elemental sulphur in a quartz tube sealed under vacuum. For the toxicity test of a bioluminescent strain ofE. coliwe used concentrations from 1 to 0.0001 g L−1and also studied fresh suspensions and suspensions left for 24 h. The strongest toxic effect was observed in freshly prepared water solutions where the luminescence of bacteria decreased by more than 75%. When saline solution was substituted for water or when the solutions were stored for 24 h it resulted in a considerable decrease in the TiS3antibacterial effect. The toxicity of TiS3in water exceeded the toxicity of the reference TiO2nanoparticles, though when saline solution was used instead of water the opposite results were observed. In addition, we did not find a relationship between the antibacterial activity of water suspensions of nanoribbons and the stability of their colloidal systems, which indicates an insignificant contribution to the toxicity of aggregation processes. In 0.9% NaCl solution suspensions, toxicity increased in proportion to the increase in the zeta potential. We suppose that the noted specificity of toxicity is associated with the emission of hydrogen sulphide molecules from the surface of nanoribbons, which, depending on the concentration, can either decrease or increase oxidative stress, which is considered the key mechanism of nanomaterial cytotoxicity. However, the exact underlying mechanisms need further investigation. Thus, we have shown an important role of the dispersion medium and the period of storage in the antibacterial activity of TiS3nanoribbons. Our results could be used in nanotoxicological studies of other two-dimensional nanomaterials, and for the development of novel antibacterial substances and other biomedical applications of this two-dimensional material.