There is a need for the development of antifouling materials to resist adsorption of biomacromolecules. Here we describe the preparation of a novel zwitterionic block copolymer with the potential to prevent or delay the formation of microbial biofilms. The block copolymer comprised a zwitterionic (hydrophilic) section of alternating glutamic acid (negatively charged) and lysine (positively charged) units and a hydrophobic polystyrene section. Cryo-TEM and dynamic-light-scattering (DLS) results showed that, on average, the block copolymer self-assembled into 7-nm-diameter micelles in aqueous solutions (0 to 100 mM NaCl, pH 6). Quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and contact angle measurements demonstrated that the block copolymer self-assembled into a brush-like monolayer on polystyrene surfaces. The brush-like monolayer produced from a 100 mg/L block copolymer solution exhibited an average distance, d, of approximately 4-8 nm between each block copolymer molecule (center to center). Once the brush-like monolayer self-assembled, it reduced EPS adsorption onto the polystyrene surface by ∼70% (mass), reduced the rate of bacterial attachment by >80%, and inhibited the development of thick biofilms. QCM-D results revealed that the EPS molecules penetrate between the chains of the brush and adsorb onto the polystyrene surface. Additionally, AFM analyses showed that the brush-like monolayer prevents the adhesion of large (> d) hydrophilic colloids onto the surface via hydration repulsion; however, molecules or colloids small enough to fit between the brush polymers (< d) were able to be adsorbed onto the surface via van der Waals interactions. Overall, we found that the penetration of extracellular organelles, as well as biopolymers through the brush, is critical for the failure of the antifouling coating, and likely could be prevented through tuning of the brush density. Stability and biofilm development testing on multiple surfaces (polypropylene, glass, and stainless steel) support practical applications of this novel block copolymer.