Hydrogel scaffolds are important materials in tissue engineering, and their characterization is essential to determine potential biomedical applications according to their mechanical and structural behavior. In this work, silk fibroin hydrogels were synthesized by two different methods (vortex and sonication), and agarose hydrogels were also obtained for comparison purposes. Samples were characterized by scanning electron microscopy, infrared analysis, thermo-gravimetrical analysis, confined compression test, and rheological test. The results indicate that nanofibers can be obtained via both silk fibroin and agarose hydrogels. The mechanical tests showed that the Young’s modulus is similar to those found in the literature, with the highest value for agarose hydrogels. All the hydrogels showed a shear-thinning behavior. Additionally, the MTT test revealed that silk fibroin hydrogels had low cytotoxicity in THP-1 and HEK-293 cells, whereas the agarose hydrogels showed high toxicity for the THP-1 cell line. The results indicate that silk fibroin hydrogels obtained from a Colombian silkworm hybrid are suitable for the development of scaffolds, with potential applications in tissue engineering.