This review discusses the latest research on spider silk and silk-based materials in biomedical and tissue engineering, focusing on musculoskeletal tissues, including skin regeneration, bone and cartilage repair, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Spider silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Silk-based biomaterials can mimic tissue structure and promote regeneration. Silk proteins are important in tissue-on-chip and organ-on-chip technologies and micro devices for artificial tissue and organ engineering. Silk has potential for controlled drug delivery at the target destination. However, challenges remain, including clinical trials. Spider silk is composed of spidroin proteins, which determine its hierarchical structure. The primary amino acids in spider silk are glycine, alanine, and serine. The strength of spider silk is highly dependent on the size and orientation of the nanocrystals. The microstructure of silk fibers is semi-crystalline, with a crystalline and amorphous phase. The nanocrystalline phase is a result of the specific polypeptide secondary structure. Silk fibers have excellent mechanical properties, including high strength and toughness. Spider silk has been used in various applications, including tissue engineering, and has been used for centuries in different applications. Natural spider silk is produced by spiders, which are cold-blooded organisms that can spin silk fibers with very high mechanical strength and toughness. The temperature in their environment affects the speed of spinning of the web, and thus its mechanical and structural properties. Silk varies widely in composition, depending on the specific source. Spiders produce seven silk types, thanks to the various silk glands located at the rear end of the abdomen. These types have different properties depending on whether they serve as a shelter, a means of catching prey, part of the love game, or as a particular thread the spider uses to escape in case of danger. Recombinant production of spider silk involves the extraction of spider silk proteins in the form of powder. This technique allows for the combination of spider silk proteins with various materials to create fibers with different mechanical and structural properties. Although using expression systems makes recombinant production cost-effective, the process of purifying spider silk protein powder is both time-consuming and expensive. Biotechnological production has opened new approaches to produce spider silk proteins from other sources like bacteria, plants, yeasts, cells, or animals, to provide cost-efficient and stable fabrication. Spider silk structures have been studied for different biomaterial applications, especially biological response, physicochemical characterization, and parameters that determine the final coating properties. These coatings can be customized from aspects of different properties aiming to support better scaffolds in tissue engineering and natural-based materials as coatings on implants, but also for the development of biosensors and to serve in surface functionalization for bioactive materials. The design of new thin films based on spider silk showed possibilities of tailoring morphologies and hydrophobicity, as veryThis review discusses the latest research on spider silk and silk-based materials in biomedical and tissue engineering, focusing on musculoskeletal tissues, including skin regeneration, bone and cartilage repair, ligaments, muscle tissue, peripheral nerves, and artificial blood vessels. Spider silk is one of the toughest natural materials, with high strain at failure and mechanical strength. Silk-based biomaterials can mimic tissue structure and promote regeneration. Silk proteins are important in tissue-on-chip and organ-on-chip technologies and micro devices for artificial tissue and organ engineering. Silk has potential for controlled drug delivery at the target destination. However, challenges remain, including clinical trials. Spider silk is composed of spidroin proteins, which determine its hierarchical structure. The primary amino acids in spider silk are glycine, alanine, and serine. The strength of spider silk is highly dependent on the size and orientation of the nanocrystals. The microstructure of silk fibers is semi-crystalline, with a crystalline and amorphous phase. The nanocrystalline phase is a result of the specific polypeptide secondary structure. Silk fibers have excellent mechanical properties, including high strength and toughness. Spider silk has been used in various applications, including tissue engineering, and has been used for centuries in different applications. Natural spider silk is produced by spiders, which are cold-blooded organisms that can spin silk fibers with very high mechanical strength and toughness. The temperature in their environment affects the speed of spinning of the web, and thus its mechanical and structural properties. Silk varies widely in composition, depending on the specific source. Spiders produce seven silk types, thanks to the various silk glands located at the rear end of the abdomen. These types have different properties depending on whether they serve as a shelter, a means of catching prey, part of the love game, or as a particular thread the spider uses to escape in case of danger. Recombinant production of spider silk involves the extraction of spider silk proteins in the form of powder. This technique allows for the combination of spider silk proteins with various materials to create fibers with different mechanical and structural properties. Although using expression systems makes recombinant production cost-effective, the process of purifying spider silk protein powder is both time-consuming and expensive. Biotechnological production has opened new approaches to produce spider silk proteins from other sources like bacteria, plants, yeasts, cells, or animals, to provide cost-efficient and stable fabrication. Spider silk structures have been studied for different biomaterial applications, especially biological response, physicochemical characterization, and parameters that determine the final coating properties. These coatings can be customized from aspects of different properties aiming to support better scaffolds in tissue engineering and natural-based materials as coatings on implants, but also for the development of biosensors and to serve in surface functionalization for bioactive materials. The design of new thin films based on spider silk showed possibilities of tailoring morphologies and hydrophobicity, as very