Doping nanoparticles represents a strategy for modulating the energy levels and surface states of nanocrystals (NCs), thereby enhancing their efficiency and mitigating toxicity. Thus, we herein focus on the successful synthesis of pure and gold (Au)-doped zinc oxide (ZnO) nanocrystals (NCs), investigating their physical−chemical properties and evaluating their applicability and toxicity through in vitro and in vivo assessments. The optical, structural, and photocatalytic characteristics of these NCs were scrutinized by using optical absorption (OA), X-ray diffraction (XRD), and methylene blue degradation, respectively. The formation and doping of the NCs were corroborated by the XRD and OA results. While the introduction of Au as a dopant did induce changes in the phase and size of ZnO, a high concentration of Au ions in ZnO led to a reduction in their photocatalytic activity. This demonstrated a restricted antibacterial efficacy against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Remarkably, Au-doped counterparts exhibited enhanced biocompatibility in comparison to ZnO, as evidenced in both in vitro (murine macrophage cells) and in vivo (Drosophila melanogaster) studies. Furthermore, confocal microscopy images showed a high luminescence of Au-doped ZnO NCs in vivo. Thus, this study underscores the potential of Au doping of ZnO NCs as a promising technique to enhance material properties and increase biocompatibility.