Recent progress in biointerface research has highlighted the role of antifouling functionalizable coatings in the development of advanced biosensors for point-of-care bioanalytical and biomedical applications dealing with real-world complex samples. The resistance to nonspecific adsorption promotes the biorecognition performance and overall increases the reliability and specificity of the analysis. However, the process of modification with biorecognition elements (so-called functionalization) may influence the resulting antifouling properties. The extent of these effects concerning both functionalization procedures potentially changing the surface architecture and properties, and the physicochemical properties of anchored biorecognition elements, remains unclear and has not been summarized in the literature yet. This critical review summarizes these key functionalization aspects with respect to diverse antifouling architectures showing low or ultra-low fouling quantitative characteristics in complex biological media such as bodily fluids or raw food samples. The subsequent discussion focuses on the impact of functionalization on fouling resistance. Furthermore, this review discusses some of the drawbacks of available surface sensitive characterization methods and highlights the importance of suitable assessment of the resistance to fouling.