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Since its inception, the field of tissue engineering has sought to rebuild the complexity of normal tissues by seeding cells onto scaffolds to support the formation of new tissue. Recently, nanofibers have gained increasing attention because these biomaterials have unique properties and are able to interface with cells at the same scale as native extracellular matrix fibrils. New fabrication technologies provide novel ways to control the nanoscale structure and properties of biomaterials, which is advantageous in the engineering of tissues for in vitro study and in vivo applications in regenerative medicine. This chapter explores the properties of nanofiber biomaterials (diameters <500 nm) and examines the specific advantages relative to other scaffold materials. This includes nanofibers from biopolymers as well as synthetic polymers, with consideration of relative advantages and disadvantages. A range of fabrication strategies is discussed that span from fiber spinning techniques, to phase separation in bulk, to directed and self-assembly. Insight is provided as to how synthetic polymers and biopolymers are used to make these nanofibers and the specific molecular structures that impart the unique mechanical, electrical, chemical, and biological properties. Analysis of these nanofiber biomaterialsrequires characterization techniques that are able to probe at the nanometer, micrometer, and macroscales. Examples are provided using optical microscopy, electron microscopy, scanning probe microscopy, mechanical characterization, and as sessment of biocompatibility and biodegradation.Finally, nanofiber biomaterials have wide applications in tissue engineering; here we focus on representative examples in cardiac, musculoskeletal, ophthalmic, and neural tissue engineering.