While environmental biofilms have recently been implicated as a potential major sink for nanoparticles (NPs), the mechanisms of interactions remain largely unknown. Polysaccharides are a common component of biofilm extracellular polymeric substances (EPS) and an initial point of contact for NPs in early NP–biofilm interactions. In this study, the significance of polysaccharide coatings on the deposition of hematite and silica NPs was examined by quartz crystal microgravimetry (QCM) and in-depth characterization of surface properties. NP deposition was shown to be largely governed by electrostatic forces. However, bulk surface zeta potential values of the tested polysaccharide-coated surfaces were not sufficient in describing the varying extent of NP deposition. Surface charge density and distribution both appeared to contribute to different NP deposition behaviors. These results suggest that nanometer to micrometer spatial characterization of biofilm surface properties, including chemical composition and charge, is necessary to improve our understanding of NP–biofilm interactions.