Mesenchymal stem or stromal cells (MSC) contribute in vivo to wound repair and can be utilized for tissue regeneration. In contrast, fibroblasts may contribute to scar formation and may even hamper functional regeneration. Depending on the clinical application, MSC are sometimes attached to a scaffold to maintain the cells in the area of regeneration. We therefore screened for proteins that allow a preferential binding of MSC and avoid strong adherence of fibroblasts. The human MSC were isolated from bone marrow (bmMSC) or term placenta (pMSC). Synovial fibroblasts (SF) and dermal fibroblasts (DF) served as controls. In the first set of experiments, binding of bmMSC and SF to extracellular matrix (ECM) proteins was investigated by multiple substrate array (MSA®). From MSA® protein analyses 57 peptides with potential MSC-binding sites were selected and the binding of the cells to these peptides was determined. We report that MSC differ from fibroblasts in their binding to proteins of the extracellular matrix. MSC bind with higher efficiency to laminin-111, collagens-I, -III, and -IV and tenascin-C compared to fibroblasts, while both cell types bind with high efficiency to fibronectin, vitronectin, and laminin-511. We conclude that overall MSC seem less selective with respect to binding extracellular matrix components compared to fibroblasts, and fibroblasts attach to fewer proteins and peptides.