Polymer brushes, in which polymers are end-tethered densely to a grafting surface, are commonly proposed for use as stealth coatings for various biomaterials. However, although their use has received considerable attention, a mechanistic understanding of the impact of brush properties on protein adsorption and unfolding remains elusive. We investigated the effect of the grafting density of poly(ethylene glycol) (PEG) brushes on the interactions of the brush with fibronectin (FN) using high-throughput single-molecule tracking methods, which directly measure protein adsorption and unfolding within the brush. We observed that, as grafting density increased, the rate of FN adsorption decreased; however, surface-adsorbed FN unfolded more readily, and unfolded molecules were retained on the surface for longer residence times relative to those of folded molecules. These results, which are critical for the rational design of PEG brushes, suggest that there is a critical balance between protein adsorption and conformation that underlies the utility of such brushes in physiological environments.