Sulfidized nanoscale zero-valent iron (S-NZVI) has been increasingly applied in environmental applications for water remediation. Therefore, the potential ecological and environmental risks of S-NZVI should be considered. This paper investigated the differences in the toxicity of six different S-NZVIs to Escherichia coli (E. coli) by focusing on the two sulfidized procedures (one-step method, two-step method) and three sulfur precursors (Na₂S, Na₂S₂O₄, Na₂S₂O₃). The experimental results show that the toxicity outcomes of the six S-NZVIs are significantly associated with the synthesis method since both sulfidized procedures and sulfur precursors can affect their features. These physicochemical properties, such as sulfur content, sulfur species, and zeta potential, play a crucial role in affecting the electron transfer efficiency and the attachment of materials with microorganisms. Consequently, this results in differing concentrations of extracellular reactive oxygen species (ROS) and intracellular ROS, particularly extracellular ·OH, which causes oxidative stress in E. coli and thus accounts for the variation in toxicity of the six S-NZVIs. Specifically, S-NZVI_two-Na₂S and S-NZVI_two-Na₂S₂O₃ exhibit higher toxicity than S-NZVI_one-Na₂S and S-NZVI_one-Na₂S₂O₃, but S-NZVI_one-Na₂S₂O₄ is more toxic than S-NZVI_two-Na₂S₂O₄. This is attributable to the abundance of S²⁻ and S₂²⁻ on the surface, which accelerates the electron transfer to produce more ·OH. Furthermore, the higher zeta potential of S-NZVI_one-Na₂S₂O₃ results in distinct attachment to the surface of E. coli compared to other S-NZVIs, ultimately decreasing its level of toxicity. By comparing the toxicity of six S-NZVIs, this study enhances understanding of the correlation between the physicochemical properties of S-NZVI and the toxic mechanism. In addition, it provides a reference point for a comprehensive evaluation of the environmental risks related to S-NZVI.