Visible-light driven photosensitizers have been recognized for their potential utility for various applications especially in nanomedicine. The aim of this study is to investigate a new strategy to use Cu-substituted ZnOnanoassemblies for induction of photodynamic effect under visible light irradiation. Here, we report the synthesis of Cu substituted ZnO nanoassemblies (Cu-ZnO NAs) with optimized Cu concentration, required for enhanced photosensitive performance for sustained antibacterial and anticancer activity under dark as well as visible-light irradiation conditions. It is noted that the substitution of Cu ions in ZnO NAs remarkably improves its absorption properties and charge separation efficiency as well as reactive oxygen species (ROS) level that make it more appropriate for photodynamic therapy under visible light irradiation for killing of bacterial and cancerous cells. The generated ROS causes a significant decrease in cell viability as well as mitochondrialmembrane potential. Moreover, Cu-ZnO NAs show ROS induced cellular apoptosis, DNA fragmentation, and depolarization of mitochondrial membrane and cell cycle arrest in G1 phase. These characteristics enable the use of these NAs as a photosensitizer in cancer therapy.