Polyelectrolyte multilayer films assembled from a hydrophobic N-alkylated polyethylenimine and a hydrophilic polyacrylate were discovered to exhibit strong antifouling, as well as antimicrobial, activities. Surfaces coated with these layer-by-layer (LbL) films, which range from 6 to 10 bilayers (up to 45 nm in thickness), adsorbed up to 20 times less protein from blood plasma than the uncoated controls. The dependence of the antifouling activity on the nature of the polycation, as well as on assembly conditions and the number of layers in the LbL films, was investigated. Changing the hydrophobicity of the polycation altered the surface composition and the resistance to protein adsorption of the LbL films. Importantly, this resistance was greater for coated surfaces with the polyanion on top; for these films, the average zeta potential pointed to a near neutral surface charge, thus, presumably minimizing their electrostatic interactions with the protein. The film surface exhibited a large contact angle hysteresis, indicating a heterogeneous topology likely due to the existence of hydrophobic-hydrophilic regions on the surface. Scanning electron micrographs of the film surface revealed the existence of nanoscale domains. We hypothesize that the existence of hydrophobic/hydrophilic nanodomains, as well as surface charge neutrality, contributes to the LbL film's resistance to protein adsorption.