Nanoparticles (NPs) are widely studied for biomedical applications. Generally, NPs and NP-based materials should be effectively sterilized without altering their structure and function before clinical translation can be considered. However, research on industrially scalable sterilization methods of nanoparticles is still scarce. Therefore, we explored if treatment of gelatin nanoparticles (GNPs) using supercritical carbon dioxide (scCO2) resulted in effective inactivation of Staphylococcus aureus as frequent pathogen causing infections. After scCO2-treatment the morphology, size and surface charge of GNPs remained unchanged, although a slight decrease in crosslinking density was observed. ScCO2 successfully inactivated Staphylococcus aureus in GNPs, as evidenced by a 12-log reduction in bacterial burden. However, cytocompatibility of GNPs was lower after scCO2 treatment, indicating that the process still requires further optimization. Nevertheless, the work presented herein highlights the strong potential of scCO2 for inactivation of pathogens in nanoparticles.