Silk fibroin (SF) from silkworms has special qualities, and these qualities have drawn a lot of interest lately in SF-based hydrogels for a range of biological applications. However, because there is a dearth of naïve silk materials to collect and prepare, research on the SF-based hydrogels isolated from spider silks has been rather limited. Thus, this study compared the microstructural properties of silk fibroin (SF) hydrogel scaffold, which was taken from the cocoon of the insect silkworm Bombyx mori, with those of hydrogel scaffolds derived from two types of woven silk glands in the orb-web spider Trichonephila clavata: the major ampullate gland (MAG) and the tubuliform gland (TG). The SF hydrogel, which is stabilized by connected SF fibers, has a loose top structure, high porosity, and translucency, according to our FESEM investigation. While the TG hydrogel showed greater porosity, ridge-like or wall-like structures, and stable biocapacity generated by physical cross-linking, the MAG hydrogel showed even higher porosity, elongated fibrous structures, and superior mechanical properties. It is anticipated that the unique microstructural properties of MAG and TG hydrogels will be advantageous when choosing customized substrates to support particular cell types for tissue engineering and regenerative medicine applications.