The synthesis of starch-based physical hydrogels in combination with chitosan and polyvinyl alcohol, and their potential co-application with chitosan nanoparticles was evaluated. The potential of starch-chitosan hydrogel obtained by physical/chemical method for tissue engineering uses was also studied in a mouse wound healing model. Although the microscopical structure of each synthesized hydrogel suggests a possible biological application, starch-polyvinyl alcohol hydrogel exhibited rigid behavior with minor channel diameters, a lower swelling rate (less than 300%), and negatively affected cell viability in a cytotoxicity assay. Starch-chitosan hydrogel obtained by chemical crosslinking with glutaraldehyde demonstrated the higher swelling rate (about 1100%), cell viability values over 80%, and a homogeneous tri-dimensional structure; along with an excellent interaction with chitosan nanoparticles. This type of hydrogel was selected for an in vivo experiment, showing significant differences in wound healing process against a non-treated control, in terms of inflammation, exudate production and tissue recovering.