Silver nanoparticle (Ag NP) dissolution, or ionization from Ag(0) to Ag+, is an important determinant of the nanoparticles' toxicity as silver ions are considered to be a major contributor to Ag NP cytotoxicity. In this work, we characterize ion dissolution from Ag NPs using a selective and dynamic technique, Ag+-selective electrodes (ISEs) with ionophore-doped fluorous sensing membranes. We examined dissolution of various concentrations of Ag NPs (0.3, 3, and 15 μg mL−1) in water and bacterial growth medium in real-time. A decrease in the concentration of free Ag+ was observed as a result of complexation with components of the growth medium. Overall, a greater percentage of the nanoparticles dissolve in growth medium than water (28% vs. 13%). Individual chemical components of the growth medium were examined for their complexation capability, and it was determined that ammonia-silver complexes are the predominant species of dissolved Ag+, with 8.9% occurring as AgNH3+, 87.8% occurring as Ag(NH3)2+, and only 3.3% occurring as free Ag+. After characterizing Ag NP dissolution in growth medium, the viability and growth of Shewanella oneidensis, a ubiquitous beneficial bacterium, were monitored upon exposure to the known in situ levels of Ag+ and Ag NPs. Ag+ and Ag NPs both caused a dose-dependent decrease in bacterial viability and growth rate, though the growth and viability changes upon Ag NP exposure did not correlate with the ISE-measured Ag+. Using ISEs to monitor Ag NP dissolution in the presence of S. oneidensis revealed that the presence of the organisms influences the nanoparticle dissolution profile, a result not previously reported that has significant implications for understanding nanotoxicity. This work lays the foundation for the use of fluorous-phase ISEs as an in situ nanoparticle characterization tool, addressing a critical technology gap in the field of nanoparticle toxicology.