This research presents a straightforward, effective, and eco-friendly method for the production of silver nanoparticles (AgNPs) and copper oxide nanoparticles (CuONPs) using the dried fruit of Amomum subulatum as a reducing, stabilizing, and capping agent. The formation of AgNPs and CuONPs is supported by the presence of a surface plasmon resonance band (SPR) at 440 nm for AgNPs and 245 nm for CuONPs. Additionally, the identification of specific biomolecules responsible for the synthesis of AgNPs and CuONPs was confirmed through FTIR spectra analysis. The Transmission electron microscope (TEM) images demonstrated that AgNPs and CuONPs had spherical shapes, with mean particle diameters of 20.6 nm and 24.7 nm, respectively. X-ray diffraction and selected area electron diffraction (SAED) analyses provided evidence of the crystalline nature of the synthesized AgNPs and CuONPs. Additionally, the presence of silver and copper elements was observed through energy-dispersive X-ray spectroscopy (EDS) analysis. Furthermore, the antibacterial activity of AgNPs was found to be superior to that of CuONPs against human pathogens such as Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The cytotoxic activity of the biosynthesized nanoparticles (NPs) was evaluated in vitro against human cervical cells (HeLa) and human breast cells (MCF-7). In MCF-7 cells, the IC50 value for AgNPs was estimated to be 39.79 µg/mL, while that of CuONPs was 83.89 µg/mL. In HeLa cells, the IC50 value for AgNPs was 45.5 µg/mL, and for CuONPs, it was 97.07 µg/mL. For the first time, an eco-friendly method for the synthesis of AgNPs and CuONPs from fruit extract of Amomum subulatum has been discussed along with their comparative evaluation study. These results highlight the promising applications of the eco-friendly synthesized AgNPs and CuONPs as effective agents against microbial infections and potential candidates for cancer therapy.