Silk is a naturally derived polymer found in various species of spiders and insects. It primarily comprises two important proteins, namely, silk fibroin (SF) and silk sericin (SS). In recent periods, silk protein-mediated nanoparticles have gathered remarkable devotion in the biomedical arena owing to their versatile properties, which include biocompatibility, ease of processing, biodegradability, excellent mechanical strength, surface modification capability, non-immunogenicity, unique biochemical nature, etc. Therefore, this book chapter delivers a summary of recent advancements in the silk protein nanoparticle-mediated biomedical applications. At first, it provides a concise introduction to silk proteins, silk protein-mediated nanoformulations, and the role of silk protein nanoparticles in wound healing plus tissue engineering. Furthermore, the potential of silk protein nanoparticles for gene delivery and ribonucleic acid (RNA) therapeutics is highlighted. The applications of silk protein nanoparticles in regenerative medicine, antibacterial, and antioxidant therapies are also discussed. Finally, the preference for silk proteins in bio/imaging and diagnostic technologies is described. Current challenges and prospects in the biomedical applications of silk protein nanoparticles are addressed, highlighting their potential to revolutionize the biomedical sector. Overall, silk protein nanoparticles have the aptitude to significantly expand biomedical research in the upcoming days.