In regenerative medicine, extracellular matrix (ECM)‐inspired materials are currently being explored to imitate mechanotransduction pathways and control cell fate. In musculoskeletal tissue regeneration, enhancing mechano‐biological signals require biomaterials that are both biocompatible and viscoelastic and can retain water content. Herein, based on these requirements, various polyvinyl alcohol (PVA)‐based composite hydrogels, reinforced by polyhydroxy butyrate (PHB) nanofibers, are proposed to differentiate equine adipose‐derived stem cells for musculoskeletal regeneration. To study the role of fiber embedding in improving scaffold properties, different nanofiber assemblies, including chopped short ones with random orientation (PVAS), single‐layer (PVAL1), and double‐layer membranes (PVAL2) are positioned into the PVA matrix. PHB reinforcements negatively affect swelling and positively enhanced phase transition temperatures and crystallinity of PVA hydrogel. According to mechanical analysis results, compositing with PHB nanofibrous layers strengthen the PVA matrix due to some restrictions on PVA chain mobility. Gene expression investigations also reveal that higher matrix stiffness after layering with two PHB membranes (PVAL2) promotes osteogenesis, while the random addition of short‐chapped fibers (PVAS) facilitate tenogenic differentiation. As a consequence of the findings, fiber placement is crucial to the mechanical properties of composite hydrogels that ultimately control musculoskeletal differentiation signals through mechanosensing pathways.