In recent times, notable advancement has been made in the field of electrospinning for the fabrication of numerous types of nanofiber scaffolds. Due to the ultrathin fiber diameter, electrospun nanofiber scaffolds are considered to be an operational delivery system for biomolecules, genes, as well as drugs due to the high specific surface area and stereological porous structure. Here, we introduce some of methods for the integration of drugs and biomolecules within electrospun nanofiber scaffolds, such as blending, surface modification, coaxial process, and emulsion methods. Then, we describe some important biomedical applications of nanofibers in drug delivery systems along with their suitable examples in transdermal systems and wound dressings, cancer therapy, growth factor delivery, nucleic acid delivery, and stem cell delivery.