Sulfidation of metallic nanoparticles such as silver nanoparticles (AgNPs) released to the environment may be an important detoxification mechanism. Two types of AgNPs—an engineered polydisperse and aggregated AgNP powder, and a laboratory-synthesized, relatively monodisperse AgNP aqueous dispersion—were studied. The particles were sulfidized to varying degrees and characterized to determine the effect of initial AgNP polydispersity and aggregation state on AgNP sulfidation, and then exposed to Escherichia coli to determine if the degree of sulfidation of pristine AgNPs affects growth inhibition of bacteria. The extent of sulfidation was found to depend on the HS–/Ag ratio. However, for the same reaction times, the more monodisperse particles were fully transformed to Ag2S, and the polydisperse, aggregated particles were not fully sulfidized, thus preserving the toxic potential of Ag0 in the aggregates. A higher Ag2S:Ag0 ratio in the sulfidized nanoparticles resulted in less growth inhibition of E. coli over 6 h of exposure. These results suggest that the initial properties of AgNPs can affect sulfidation products, which in turn affect microbial growth inhibition, and that these properties should be considered in assessing the environmental impact of AgNPs.