To determine whether the use of multiple layers of acellular bladder matrix (ABM) is more suitable for the treatment of abdominal wall hernia than a single layered ABM. The feasibility, biocompatibility and mechanical properties of both materials were assessed and compared. Biocompatibility testing was performed on 4 and 1 layered ABM. The matrices were used to repair an abdominal hernia model in 24 rabbits. The animals were followed for up to 3 months. Immediately after euthanasia, the implant site was inspected and samples were retrieved for histology, scanning electron microscopy and biomechanical studies. Both acellular biomaterials demonstrated excellent biocompatibility. At the time of retrieval, there was no evidence of infection. The matrices demonstrated biomechanical properties comparable to native tissue. Three hernias (25%) were found in the single layer ABM group and only 1 hernia (8%) was found in the 4 layer ABM group. Histologically, the matrix structure was intact and the cell density within the matrices decreased with time. The dominant cell type present within the matrices shifted from lymphocytes to fibroblasts over time. Both ABMs maintained adequate strength over time when used for hernia repair, and there was an extremely low incidence of adhesion formation. The single layer ABM showed enhanced cellular integration, while the 4 layer ABM reduced hernia formation. Either of these matrices may be useful as an off-the-shelf biomaterial for patients requiring fascial repair.