Gold nanoclusters emitting in the second biological window (NIR-II-AuNCs) have gained significant interest for their potential in deep-tissue bioimaging and biosensing applications due to the partial transparency and reduced autofluorescence of tissues in this spectral range. However, the limited understanding of how the biological environment affects their luminescent properties might hinder their use in bioimaging and biosensing. In this study, we investigated the emission properties of NIR-II-AuNCs when interacting and internalizing into live cells including macrophages, fibroblasts, and cancer cell lines, revealing substantial alterations in their luminescence. A systematic comparison between control and in vitro experiments concluded that the disruption of surface ligands is the main factor responsible for these alterations. NIR-II-AuNCs within cellular environments may also be influenced by other interactions, including aggregation or complexation with proteins. Furthermore, we also corroborated these spectroscopic modifications at the in vivo level, providing additional evidence of the environmental sensitivity of NIR-II-AuNCs. The results obtained in this study contribute to a deeper understanding of the luminescence mechanisms of NIR-II-AuNCs in biological environments in cells and in living tissues and are crucial for their optimization as reliable tools in biological environment for in vitro and in vivo imaging and diagnostics.