The preparation and study of three-dimensional functional skin substitutes has been the focus of intense research for several decades. Dermal substitutes are now commonly used in medical practice for a variety of applications. Here, we assess the toxicity of seven selected acellular dermal matrix materials to establish their potential for use in future three-dimensional skin substitute studies. The cytotoxicity of acellular dermis (of Allo- and Xenograft origin) prepared in our lab and biomaterials based on collagen and hyaluronic acid (Coladerm H and Coladerm H-L) were compared to that seen in three commercially available products (Xe-Derma, AlloDerm and Xeno-Impl). Murine fibroblasts NIH-3T3 and human dermal fibroblasts were used in cytotoxicity tests, with any resultant cytotoxic effects caused by the seven tested dermal scaffolds visualised using an inverted microscope system and confirmed in parallel using colorimetric MTT cell proliferation assays. While most of the dermal substitutes did not demonstrate a cytotoxic effect on our two cell types, Xeno and Xeno-Impl scaffolds clearly did. The cytotoxic effect of acellular Xeno dermal matrix could essentially be removed through a regime of multiple washes, but we were unable to remove the cytotoxic effect of Xeno-Impl. Thus, Xeno-Impl alone has been excluded from our future work on preparation of 3D skin substitutes.