Electrospinning is a versatile technique providing highly tunable nanofibrous nonwovens. Many biomedical applications have been developed for nanofibers, among which the production of antimicrobial mats stands out. The production of scaffolds for tissue engineering, fibers for controlled drug release, or active wound dressings are active fields of research exploiting the possibilities offered by electrospun materials. The fabrication of materials for active food packaging or membranes for environmental applications is also reviewed. We attempted to give an overview of the most recent literature related with applications in which nanofibers get in contact with living cells and develop a nano-bio interface.