A study was conducted to investigate the role of nanoparticle (NP) surface functionalization/charge on their uptake by biofilms. Biofilms, bacterial colonies attached to surfaces via extracellular polymers, are effective at removing suspended nanomaterials from the aqueous phase. However, the mechanisms regulating particle uptake are unknown. Here, it was shown that the mechanism was strongly dependent on the nanoparticle surface ionization, and not the core composition of the NP. Uptake experiments were conducted using laboratory-cultured biofilms. The biofilms were incubated in the presence of fluorescent polystyrene NPs with either negatively-charged surfaces (i.e. functionalized with sulfated (SO(4) (-)-NP) or carboxylated (COO(-)-NP) groups) or positively-charged surfaces (functionalized with primary amines, Amine-P). Particles with negatively-charged sulfated surfaces associated most strongly to biofilms across all experimental conditions. Associations of positively-charged amine particles with biofilms were greatest at high ionic conditions resembling those of seawater, but were sensitive to changes in ionic strength. Sorption of COO(-)-NPs was lowest, relative to other particle types, and was not sensitive to ionic strength. The results of this study support an emerging precedent that biofilms may be an effective player in the binding and sequestration of nanoparticles in aqueous systems.