The majority of bacteria in the natural environment exist as biofilms that linger on surfaces even under rigorous antibiotic treatment. Biofilms are enclosed within an exopolymeric substance (EPS) that restricts the diffusion of large and small molecules, an underlying factor to antibiotic resistance in biofilms. The aim of this study is to gain insight into the forces involved in the interaction of alpha-helical cationic antimicrobial peptides and the Pseudomonas aeruginosa biofilm EPS alginate. These peptides are unique in their ability to permeate bacterial cells by spontaneously inserting into the membrane to form pores likely consisting of alpha-helical bundles. However, the presence of alginate alone is often sufficient to induce, perhaps prematurely, alpha-helix conformation in these peptides and compete for their interaction with membranes. Our results suggest that the reduced transport of cationic antimicrobial peptides in the presence of alginate involves a certain balance between hydrophobic interactions and electrostatic interactions.