The formation of 3D electrospun mat structures from alginate–polyethylene oxide (PEO) solution blends is reported. These unique architectures expand the capabilities of traditional electrospun mats for applications such as regenerative medicine, where a scaffold can help to promote tissue growth in three dimensions. The mat structures extend off the surface of the flat collector plate without the need of any modifications in the electrospinning apparatus, are self-supported when the electric field is removed, and are composed of bundles of nanofibers. A mechanism for the unique formations is proposed, based on the fiber–fiber repulsions from surface charges on the negatively charged alginate. Furthermore, the role of the electric field in the distribution of alginate within the nanofibers is discussed. X-ray photoelectron spectroscopy is used to analyze the surface composition of the electrospun nanofiber mats and the data is related to cast films made in the absence of the electric field. Further techniques to tailor the 3D architecture and nanofiber morphology by changing the surface tension and relative humidity are also discussed.