A persistent challenge in tissue engineering is the fabrication of manipulatable scaffolds for implantation in clinical treatments and use in disease models for drug screening. Electrospinning of nanofibrous membranes is an emerging technology in artificial extracellular matrix (ECM) design that can offer precisely tunable microenvironments upon assembly into three-dimensional (3D) scaffolds that mimic the in vivo ECM structure. In this study, we report a facile and versatile strategy for preparing 3D multilayered constructs from Fe3O4/polycaprolactone (PCL)/gelatin nanofibrous membranes. This method combines membrane assembly with noncontact magnetic force to preserve the mechanical integrity and interconnectivity of the 3D scaffolds. An ordered layer structure can be achieved using a magnetic control technique through the addition of magnetic nanoparticles into the PCL/gelatin nanofibers. We first verified the magnetic properties and structures of magnetic nanofibers according to X-ray diffraction, hysteresis, scanning electron microscopy, and transmission electron microscopy. We tested the potential toxicity and osteogenic differentiation of mesenchymal stem cells seeded on the layered scaffolds. To add further functionality to the scaffolds, the membranes were coated with silver nanoparticles and shown to inhibit the growth of Escherichia coli and Staphylococcus aureus, which are responsible for most cases of infection-related implant failure. Finally, we tested the utility of magnetic membranes implanted in an animal model as a contrast agent for magnetic resonance imaging. Scaffolds formed using the presented magnetically guided fabrication strategy have the potential to mimic the structure and function of human tissues and also may be applied in disease models to study cell-cell interactions.