Background Although chemical antiseptics are the most basic measure to control wound infection and frequently come into contact with subcutaneous adipose tissue, no studies have evaluated their toxicity on adipose tissue and its cell fractions. In the present study, the effects of the following antiseptics on adipose-derived stem cells (ASC) were evaluated: Betaisodona® (povidone iodine), Lavasept®/Prontosan® (polyhexanide), Mafenide acetate, and Octenisept® (octenidinedihydrochloride). Methods Human ASCs were harvested from healthy donors. ASC viability was measured after treatment with different concentrations of antiseptics over five days. Furthermore, the effect on the proliferation, adipogenic differentiation, and apoptosis/necrosis of ASCs was analyzed. Finally, the mRNA expression of the stem cell markers CD29, CD34, CD73, CD90, and CD105 was detected. Results Octenisept® and Betaisodona® significantly reduced ASC proliferation and differentiation and led to considerable ASC necrosis. Octenisept® decreased ASC viability at the lowest concentrations tested and all stem cell markers were down-regulated by Octeniseptr® and Betaisodona®. Lavasept® and Prontosan® both led to reduced ASC viability, proliferation, differentiation, and increased apoptosis/necrosis, though the effects were less pronounced than those of Octenisept® and Betaisodona®. ASCs survived treatment with Mafenide acetate even at high concentrations, and Mafenide acetate showed minimal negative effects on their proliferation, adipogenic differentiation, cell death, and stem cell marker expression. Conclusion Mafenide acetate may be regarded as a feasible antiseptic for the treatment of wounds with exposed adipose tissue due to its low ASC toxicity. While Lavasept® and Prontosan® are possible alternatives, Octenisept® and Betaisodona® may only be used in highly diluted solutions.