Electrospinning is an economical and relatively simple method to produce continuous and uniform nanofibers from almost any synthetic and many natural polymers. Because of the high specific surface area, tunable pore size, and flexibility, the nanofibrous membranes are finding an increasingly wide range of applications. Some particular attention has been devoted to antibacterial nanofibers for applications such as wound dressings. A variety of biocides, e.g., antibiotics, quaternary ammonium salts, triclosan, biguanides, (silver, titanium dioxide, and zinc oxide) nanoparticles and chitosan have been incorporated by various techniques into nanofibers that exhibit strong antibacterial activity in standard assays. However, the small diameters of the nanofibers also mean that the incorporated biocides are often burst released once the materials are submerged in an aqueous solution. Nevertheless, several strategies, such as coresheath structure of the nanofiber, covalent bonding of the biocide on the fiber surface and adsorption of the biocide in nanostructures, can be utilized to sustain the release over several days. This review summarizes recent development in the fabrication of antibacterial nanofibers, the release profiles of the biocides and their applications in in vivo systems.