Motivated by the need to understand environmental risks posed by potentially biocidal engineered nanoparticles, the effects of silver nanoparticle (AgNP) exposure on viability in single species Pseudomonas fluorescens biofilms were determined via dye staining methods. AgNP dispersions, containing both particles and dissolved silver originating from the particles, negatively impacted biofilm viability in a dose-dependent manner. No silver treatments (up to 100 ppm AgNPs) resulted in 100% biofilm viability loss, even though these same concentrations caused complete viability loss in planktonic culture, suggesting some biofilm tolerance to AgNP toxicity. Colloidally stable AgNP suspensions exhibited greater toxicity to biofilms than corresponding particle-free supernatants containing only dissolved silver released from the particles. This distinct nanoparticle-specific toxicity was not observed for less stable, highly aggregated particles, suggesting that biofilms were protected against nanoparticle aggregate toxicity. In both the stable and highly aggregated dispersions, dissolved silver made a significant contribution to overall toxicity. Therefore, despite increased colloidal stability when humic acid adsorbed to AgNPs, the presence of humic acid mitigated the toxicity of AgNP suspensions because it bound to silver ions in solution.