The antibacterial activity of silver nanoparticles (AgNPs) is partially due to the release of Ag(+), although discerning the contribution of AgNPs vs Ag(+) is challenging due to their common co-occurrence. We discerned the toxicity of Ag(+) versus a commercially available AgNP (35.4 ± 5.1 nm, coated with amorphous carbon) by conducting antibacterial assays under anaerobic conditions that preclude Ag₀ oxidation, which is a prerequisite for Ag(+) release. These AgNPs were 20× less toxic to E. coli than Ag(+) (EC₅₀: 2.04 ± 0.07 vs 0.10 ± 0.01 mg/L), and their toxicity increased 2.3-fold after exposure to air for 0.5 h (EC₅₀: 0.87 ± 0.03 mg/L) which promoted Ag(+) release. No significant difference in Ag(+) toxicity was observed between anaerobic and aerobic conditions, which rules out oxidative stress by ROS as an important antibacterial mechanism for Ag(+). The toxicity of Ag(+) (2.94 μmol/L) was eliminated by equivalent cysteine or sulfide; the latter exceeded the solubility product equilibrium constant (K(sp)), which is conducive to silver precipitation. Equivalent chloride and phosphate concentrations also reduced Ag(+) toxicity without exceeding K(sp). Thus, some common ligands can hinder the bioavailability and mitigate the toxicity of Ag(+) at relatively low concentrations that do not induce silver precipitation. Furthermore, low concentrations of chloride (0.1 mg/L) mitigated the toxicity of Ag(+) but not that of AgNPs, suggesting that previous reports of higher AgNPs toxicity than their equivalent Ag(+) concentration might be due to the presence of common ligands that preferentially decrease the bioavailability and toxicity of Ag(+). Overall, these results show that the presence of O₂ or common ligands can differentially affect the toxicity of AgNPs vs Ag(+), and underscore the importance of water chemistry in the mode of action of AgNPs.