Background: There is an increasing demand for the clinical application of human skin substitutes (HSSs) for treating ulcers, burns and surgical wounds. Due to this increasing demand and due to the simultaneous requirement for the administration of topical antiseptic medications, there is a need to determine potential cytotoxic effects of these medications on HSSs compared with autograft skin. Objectives: To perform such an evaluation. Methods: Two different HSSs were used (autologous reconstructed epidermis on fibroblast-populated human dermis and allogeneic reconstructed epidermis on a fibroblast-populated rat collagen gel) and were compared with conventional full-thickness autograft. Twelve different antiseptics were applied topically to the stratum corneum in vitro for 24 h. The degree of cytotoxicity was analysed as detrimental changes in histology, metabolic activity (MTT assay) and RNA staining of tissue sections. Results: The antiseptic medications tested showed different degrees of cytotoxicity. Acticoat®, Aquacel Ag®, Dermacyn®, Fucidin®, 0.5% silver nitrate solution and chlorhexidine digluconate were not cytotoxic for either HSS or autograft, and can therefore be used as required. Flamazine® and zinc oxide cream resulted in moderate cytotoxicity. However, application of Betadine®, cerium-silver sulfadiazine cream, silver sulfadiazine cream with 1% acetic acid and Furacine® resulted in a substantial decrease in cell viability and a detrimental effect on tissue histology when applied to autograft and especially to HSS. Conclusions: Due to the potential cytotoxic effect of some antiseptics on HSS, it is advised that clinicians balance the cytotoxicity of the medication, its antiseptic properties and the severity of colonization in choosing which one to apply.