Polymer brushes not only represent emerging surface platforms for numerous bioanalytical and biological applications but also create advanced surface-tethered systems to mimic real-life biological processes. In particular, zwitterionic and nonionic polymer brushes have been intensively studied because of their extraordinary resistance to nonspecific adsorption of biomolecules (antifouling characteristics) as well as the ability to be functionalized with bioactive molecules. However, the relation between antifouling behavior in real-world biological media and structural changes of polymer brushes induced by surface preconditioning in different environments remains unexplored. In this work, we use multiple methods to study the structural properties of numerous brushes under variable ionic concentrations and determine the impact of these changes on resistance to fouling from undiluted blood plasma. We describe different mechanisms of swelling, depending on both the polymer brush coating properties and the environmental conditions that affect changes in both hydration levels and thickness. Using both fluorescent and surface plasmon resonance methods, we found that the antifouling behavior of these brushes is strongly dependent on the aforementioned structural changes. Moreover, preconditioning of the brush coatings (incubation at a variable salt concentration or drying) prior to biomolecule interaction may significantly improve the antifouling performance. These results suggest a new simple approach to improve the antifouling behavior of polymer brushes. In addition, the results herein enhance the understanding for improved design of antifouling and bioresponsive brushes employed in biosensor and biomimetic applications.