The mechanical properties and water permeability of hierarchical self-assembling membranes and sacs formed from oppositely charged high molecular weight hyaluronic acid (HA) and small molecule peptide amphiphiles (PAs) were studied. Techniques to make reproducible 2D planar membranes and 3D spherical sacs from these materials were developed while membrane inflation and osmotic swelling were used to quantify the mechanical properties and water permeability of these structures. It was found that incubation time and concentration of HA used had an effect on the area modulus and water permeability of the membranes. These factors also affected the kinetics of membrane growth as evidenced in SEM micrographs, which showed differences in the structure. Area modulus of membranes changed from about 6 N m⁻¹ for the lower weight percent HA system at the shortest incubation time of 3 minutes, up to 12 N m⁻¹ for the higher weight percent HA system at the longest incubation time of 60 minutes. Water permeability decreased with incubation time, but the lower weight percent HA system showed a lower water permeability when compared to the higher weight percent HA system at the same incubation time. This type of characterization and understanding of the structure–property relationships in self-assembling systems are necessary steps in both using these structures for specific applications and applying this knowledge to design new and better materials in the future.