Electrospun nanofibrous mats have recently been employed as drug reservoirs for their unique features, such as high surface-to-volume ratios and easy fabrication process. We describe herein various methods of fabricating drug- and gene-encapsulated nanofibrous meshes, which can be prepared by electrospinning. The electrospinning process of nanofibrous mats is affected by many parameters, including viscosity and ejection speeds of the polymeric solutions and the electrical potential applied to the system. Both single- and dual-nozzle systems are widely employed in the preparation of electrospun nanofibers encapsulating drugs and genes, which are usually incorporated into the electrospun mats either by physical mixing with polymeric solutions before electrospinning or by physical incorporation after electrospinning. Various strategies have been tailored to maintain the bioactivity of proteins for tissue regeneration before and after electrospinning. Nucleic acids, such as DNA and siRNA, are also incorporated into nanofibrous meshes to enhance tissue regeneration by expressing transgenes or silencing domestic genes in specific tissues. Drug- or gene-incorporated nanofibrous meshes can greatly increase tissue regeneration rates and reduce scar formation in normal and diabetic wounds. Hybrid nanofibers, with multiple cell layers or hydrogels, have also been used to improve wound healing efficiency by increasing cell infiltration.