Toxicity of nanomaterials, especially silver nanoparticles (AgNPs), is attracting increasing interests in both research and applications as the emerging of nanoscience and nanotechnology. The objective of this study was to elucidate the influences of various natural organic matter (NOM) and chelating agent, ethylenediaminetetraacetic acid (EDTA), on AgNPs stability (aggregation, dissolution) and bactericidal activity in environmentally relevant freshwater matrices. Bacterial viability, in terms of half maximal inhibitory concentration (IC₅₀) value, was adopted to judge the toxicity of AgNPs against bacteria. Our results showed that three model NOM, humic acid, bovine serum albumin and alginic acid, at concentration comparable to that of natural freshwaters did not substantially affect the bactericidal activity of AgNPs against Bacillus subtilis and Escherichia coli. Nevertheless, the combined addition of Ca²⁺ and humic acid induced severe aggregation of AgNPs, resulting in a reduced nanotoxicity. The chelating agent, EDTA, exhibited a concentration-dependent effect on AgNPs bactericidal activity: at low concentration (1 mg/L), EDTA decreased toxicity of AgNPs likely by converting the relatively high toxic dissolved Ag species into a series of low toxic Ag–EDTA complex; at high concentrations (5 and 20 mg/L), EDTA mainly acted as a synergist and increased the toxicity of AgNPs probably by reacting with divalent cations (such as Ca²⁺ and Mg²⁺) on bacterial cell membranes. Our findings have significant implications for understanding the influencing factors of AgNPs toxicity under natural freshwater conditions, which may also benefit the studies of nanotoxicity mechanisms as well as environmental impact assessments of nanomaterials.