As one of the leading causes of global mortality, cancer has prompted extensive research and development to advance efficacious drug discovery, sustained drug delivery and improved sensitivity in diagnosis. Towards these applications, nanofibers synthesized by electrospinning have exhibited great clinical potential as a biomimetic tumor microenvironment model for drug screening, a controllable platform for localized, prolonged drug release for cancer therapy, and a highly sensitive cancer diagnostic tool for capture and isolation of circulating tumor cells in the bloodstream and for detection of cancer-associated biomarkers. This review provides an overview of applied nanofiber design with focus on versatile electrospinning fabrication techniques. The influence of topographical, physical, and biochemical properties on the function of nanofiber assemblies is discussed, as well as current and foreseeable barriers to the clinical translation of applied nanofibers in the field of oncology.