Silk, a natural biopolymer, has been used clinically as suture material over thousands of years and has received much impetus for a plethora of biomedical applications in the last two decades. Silk protein isolated from both mulberry and nonmulberry silkworm varieties gained recognition as a potential biomaterial owing to its affordability and remarkable physicochemical properties. Molecular studies on the amino acid composition and conformation of silk proteins interpreted in the present review provide a critical understanding of the difference in crystallinity, hydrophobicity, and tensile strength among silkworm silk proteins. Meticulous silk fibroin (SF) isolation procedures and innovative processing techniques to fabricate gamut of two-dimensional (2D) and three-dimensional (3D) matrices including the latest 3D printed scaffolds have led SF for diverse biomedical applications. Crucial factors for clinical success of any biomaterial, including biocompatibility, immune response, and biodegradability, are discussed with particular emphasis on the lesser-known endemic nonmulberry silk varieties, which in recent years have gained considerable attention. The tunable biodegradation and bioresorbable attributes of SF enabled its use in drug delivery systems, thus proving it as an efficient and specific vehicle for controlled drug release and targeted drug delivery. Advancements in fabrication methodologies inspired biomedical researchers to develop SF-based tissue models mimicking the spatiotemporal arrangement and cellular distribution of native tissue. tissue models own a unique demand for studying tissue biology, cellular crosstalks, disease modeling, drug designing, and high throughput drug screening applications. Significant progress in silk biomaterial research has evolved into several silk-based healthcare products in the market. Insights of silk-based products assessed in the human clinical trials are presented in this review. Overall, the current review explores the paradigm of the silk structure-function relationship driving silk-based biomaterials toward tissue engineering, drug delivery systems, and tissue models.