Sequential layer-by-layer (LbL) deposition of polyelectrolytes followed by chemical cross-linking was investigated as a method to fabricate functional amphiphilic surfaces for marine biofouling prevention applications. A novel polyanion, grafted with amphiphilic perfluoroalkyl polyethylene glycol (fPEG) side chains, was synthesized and subsequently used to introduce amphiphilic character to the LbL film. The structure of the polyanion was confirmed by FTIR and NMR. Amphiphilicity of the film assembly was demonstrated by both water and hexadecane static contact angles. XPS studies of the cross-linked and annealed amphiphilic LbL films revealed the increased concentration of fPEG content at the film interface. In antifouling assays, the amphiphilic LbL films effectively prevented the adhesion of the marine bacterium Pseudomonas (NCIMB 2021).