Bacteria are the source of many bioactive compounds, including polymers with various physiological functions and the potential for medical applications. Pyomelanin fromPseudomonas aeruginosa, a nonfermenting Gram-negative bacterium, is a black–brown negatively charged extracellular polymer of homogentisic acid produced during L-tyrosine catabolism. Due to its chemical properties and the presence of active functional groups, pyomelanin is a candidate for the development of new antioxidant, antimicrobial and immunomodulatory formulations. This work aimed to obtain bacterial water-soluble (Pyosol), water-insoluble (Pyoinsol) and synthetic (sPyo) pyomelanin variants and characterize their chemical structure, thermosensitivity and biosafety in vitro and in vivo (Galleria mallonella). FTIR analysis showed that aromatic ring connections in the polymer chains were dominant in Pyosoland sPyo, whereas Pyoinsolhad fewer Car-Carlinks between rings. The differences in chemical structure influence the solubility of various forms of pyomelanins, their thermal stability and biological activity. Pyosoland Pyoinsolshowed higher biological safety than sPyo. The obtained results qualify Pyosoland Pyoinsolfor evaluation of their antimicrobial, immunomodulatory and proregenerative activities.