With the increasing number of nanotechnology applications, it is reasonable to expect nanoparticles to be ubiquitous in biofilms found in natural and engineered aquatic systems. We studied the impact of the degree of cross-linking on the deposition and diffusion of polystyrene nanoparticles (NPs) in alginate model biofilm matrices in the presence and absence of calcium cross-linkers using image correlation methods and single particle tracking. We found that cross-linking increases the viscoelasticity and hydration of the polymeric matrix and leads to structural changes that can restrict and alter the diffusive behavior of NPs, but the magnitude of the effects on diffusion depends on NP size. Nonetheless, all sizes of particles considered in the study experienced a degree of confinement and partial confinement demonstrating that diffusion in heterogeneous biofilm matrices should not be assumed to be isotropic. In bacterial biofilms, investigated using the same techniques, NP diffusion modes are dependent on biofilm age and NP functional groups and were affected by the intrinsic variability of biological systems, even when the biofilms were formed in similar conditions and by the same microorganism. Living biofilms are dynamic and active with responsive interchange between the microbial inhabitants and the biofilm structure. The results from our studies suggest that when NPs accumulate in biofilms, NPs can stress the microbes and alter gene expression of key extracellular polymeric substances production and quorum sensing systems. These changes depend on NP surface charge, gene function and biofilm age and could end up affecting biofilm architecture and metabolic efficiency. These findings elucidating the conditions that affect biofilm structure add to our knowledge of the interactions between biofilms and nanoparticles, leading to a better understanding of the fate, transport, and effect of NPs in the environment and should be useful in the development of biofilm related applications to achieve specific objectives. For example, in medicine, to eradicate biofilm infections by improving the penetration of antibiotics delivered by nanomaterials, or in environmental engineering, to determine how environmental conditions can impact the accumulation of NPs in biofilms in the environment.