Research on green production methods for metal oxide nanoparticles (NPs) is growing, with the objective to overcome the potential hazards of these chemicals for a safer environment. In this study, facile, ecofriendly synthesis of copper oxide (CuO) nanoparticles was successfully achieved using aqueous extract of Pterospermum acerifolium leaves. P. acerifolium-fabricated CuO nanoparticles were further characterized by UV-Visible spectroscopy, field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and dynamic light scattering (DLS). Plant-mediated CuO nanoparticles were found to be oval shaped and well dispersed in suspension. XPS confirmed the elemental composition of P. acerifolium-mediated copper nanoparticles as comprised purely of copper and oxygen. DLS measurements and ion release profile showed that P. acerifolium-mediated copper nanoparticles were more stable than the engineered CuO NPs. Copper oxide nanoparticles are used in many applications; therefore, their potential toxicity cannot be ignored. A comparative study was performed to investigate the bio-toxic impacts of plant-synthesized and engineered CuO nanoparticles on water flea Daphnia. Experiments were conducted to investigate the 48-h acute toxicity of engineered CuO NPs and plant-synthesized nanoparticles. Lower EC50 value 0.102 ± 0.019 mg/L was observed for engineered CuO NPs, while 0.69 ± 0.226 mg/L was observed for plant-synthesized CuO NPs. Additionally, ion release from CuO nanoparticles and 48-h accumulation of these nano CuOs in daphnids were also calculated. Our findings thus suggest that the contribution of released ions from nanoparticles and particles/ions accumulation in Daphnia needs to be interpreted with care.