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The electrospinning (ES) technique in the fabrication of biomaterials-based electrospun nanofibers (ESNFs) has risen to prominence because of its accessibility, cost-effectiveness, high production rate and diverse biomedical applications. The ESNFs have unique characteristics, such as stability and mechanical performance, high permeability, porosity, high surface area to volume ratio, and ease of functionalization. The characteristics of ESNFs can be controlled by varying either process variables or biomaterial solution properties. The active pharmaceutical agents can be introduced into ESNFs by blending, surface modification, or emulsion formation. In this chapter, in the first part, we briefly discuss the fundamental aspects of the fabrication, commonly used materials, process parameters, and characterization of ESNFs. In the second part, we discuss in detail the biomedical applications of ESNFs in drug delivery, tissue engineering, and wound healings, cancer therapy, dentistry, medical filtration, biosensing and imaging of disease.