The study of interaction mechanisms at the nanoscale between nanomaterials and microbial cells is of great importance in a wide range of research fields from health to the environment. In the latter case, due to the high occurrence of nanoparticulate systems in the environment, in-depth investigation of nanoparticle/bacteria interactions at the nanoscale is needed to better understand the full picture of natural, chemical and biological processes of the microbial biodegradation of natural or manufactured organic nanoparticles in ecosystems. In this paper, we highlight for the first time the proof of the biodegradation process of gelatin adsorbed on the surface of nanoparticles by Alteromonas macleodii extracellular proteases. Using model core–shell gold@silica@gelatin nanoparticles, the biodegradation process has been demonstrated through a novel combined multi-technique approach using mass spectrometry (ToF-SIMS, LC-ESI-MS/MS, SP-ICP-MS), photo-electron spectroscopy (XPS) and electron microscopy (SEM, TEM).