Hydrophilic nanocarriers formed by electrostatic interaction of chitosan with oppositely charged macromolecules have a high potential as vectors in biomedical and pharmaceutical applications. However, comprehensive information about the fate of such nanomaterials in biological environment is lacking. We used chitosan from both animal and fungal sources to form well-characterized chitosan-pentasodium triphosphate (TPP)//alginate nanogels suitable for comparative studies. Upon exposure of human colon cancer cells (HT29 and CaCo2), breast cancer cells (MDA-MB-231 and MCF-7), glioblastoma cells (LN229), lung cancer cells (A549), and brain-derived endothelial cells (HCEC) to chitosan-(TPP)//alginate nanogels, cell type-, nanogel dosage-, and exposure time-dependent responses are observed. Comparing chitosan-TPP//alginate nanogels prepared from either animal or fungal source in terms of nanogel formation, cell uptake, reactive oxygen species production, and metabolic cell activity, no significant differences become obvious. The results identify fungal chitosan as an alternative to animal chitosan in particular if biomedical/pharmaceutical applications are intended.