In order to evaluate the risk of engineered nanomaterials in the natural environment, one must determine their mobility, among other factors. Such determinations are difficult given that natural systems are heterogeneous and biofilms are ubiquitous in soils and waters. The interaction and diffusion of several model nanoparticles (dextrans, fluorescent microspheres, Ag nanoparticles) were studied in situ using confocal microscopy and fluorescence correlation spectroscopy in a biofilm composed of Pseudomonas fluorescens. For the most part, relative self-diffusion coefficients decreased exponentially with the square of the radius of the nanoparticle. The precise growth conditions of the biofilm resulted in a variable density of both exopolymers and microbes, which was also shown to be an important parameter controlling the diffusion of the nanoparticles. Finally, the charge of the nanoparticles appeared to be important; for a dense bacterial biofilm, a greater than predicted decrease in the self-diffusion coefficient was observed for the negatively charged nano Ag.