The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece, (2) acellular dermal replacement, Alloderm, (3) knitted poly(lactic-co-glycolic acid) (10:90)-poly(-caprolactone) (PLGA-PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [(3)H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece, Alloderm and PLGA-PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece contracted to 10% of the original size while Alloderm supported cell proliferation predominantly on the surface of the material. PLGA-PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.