Nanotechnology is one of the most dynamic research areas and the fastest-growing market. Developing eco-friendly products using available resources to acquire maximum production, better yield, and stability is a great challenge for nanotechnology. In this study, copper nanoparticles (CuNP) were synthesized via the green method using root extract of the medical plant Rhatany (Krameriasp.) as a reducing and capping agent and used to investigate the influence of microorganisms. The maximum production of CuNP was noted at 70 °C after 3 h of reaction time. The formation of nanoparticles was confirmed through UV-spectrophotometer, and the product showed an absorbance peak in the 422–430 nm range. The functional groups were observed using the FTIR technique, such as isocyanic acid attached to stabilize the nanoparticles. The spherical nature and average crystal sizes of the particle (6.16 nm) were determined using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and X-ray diffractometer (XRD) analysis. In tests with a few drug-resistant pathogenic bacteria and fungus species, CuNP showed encouraging antimicrobial efficacy. CuNP had a significant antioxidant capacity of 83.81% at 200 g/m−1. Green synthesized CuNP are cost-effective and nontoxic and can be applied in agriculture, biomedical, and other fields.