Confocal laser scanning microscopy with fluorescent markers and index matching has been used to collect three-dimensional (3D) digitized images of electrospun fiber mats and of a borosilicate glass fiber material. By embedding the fluorescent dye in either the material component (fibers) or pore space component (the index-matching fluid), acquisitions of both positive and negative images of the porous fibrous materials are demonstrated. Image analysis techniques are then applied to the 3D reconstructions of the fibrous materials to extract important morphological characteristics such as porosity, specific surface area, distributions of fiber diameter and of pore diameter, and fiber orientation distribution; the results are compared with other experimental measurements where available. The topology of the pore space is quantified for an electrospun mat for the first time using the Euler-Poincar, characteristic. Finally, a method is presented for subdividing the pore space into a network of cavities and the gates that interconnect them, by which the network structure of the pore space in these electrospun mats is determined.