Background: A suitable scaffold with adapted mechanical and biological properties for ligament tissue engineering is still missing. (2) Methods: Different scaffold configurations were characterized in terms of morphology and a mechanical response, and their interactions with two types of stem cells (Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs)) were assessed. The scaffold configurations consisted of multilayer braids with various number of silk layers (n = 1, 2, 3), and a novel composite scaffold made of a layer of copoly(lactic acid-co-(e-caprolactone)) (PLCL) embedded between two layers of silk. (3) Results: The insertion of a PLCL layer resulted in a higher porosity and better mechanical behavior compared with pure silk scaffold. The metabolic activities of both WJ-MSCs and BM-MSCs increased from day 1 to day 7 except for the three-layer silk scaffold (S3), probably due to its lower porosity. Collagen I (Col I), collagen III (Col III) and tenascin-c (TNC) were expressed by both MSCs on all scaffolds, and expression of Col I was higher than Col III and TNC. (4) Conclusions: the silk/PLCL composite scaffolds constituted the most suitable tested configuration to support MSCs migration, proliferation and tissue synthesis towards ligament tissue engineering.