The growth of material science and technology places a high importance on the creation of better processes for the synthesis of copper nanoparticles. So that, an easy, ecological, and benign process for producing copper nanoparticles (CuNPs) has been developed using candy leaf (Stevia rebaudiana) leaves aqueous extract for the first time. UV-visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), high-resolution transmission electron microscope (HR-TEM), Fourier transmission infrared (FTIR), and zeta potential were applied to demonstrate strong characterization for the biosynthesized stevia-CuNPs. The UV-visible absorbance at 575 nm of surface plasmon resonance (SPR) was 1.2. The particle size mean diameter was recorded as 362.3 nm with - 10.8 mV zeta potential. The HR-TEM scanning revealed 51.46-53.17 nm and spherical-shaped stevia-CuNPs surrounded by coat-shell proteins. The cytotoxicity and cytocompatibility activity assay revealed that stevia-CuNPs was safe in lower concentrations and had a significant cell viability reduction in higher concentrations. The produced stevia-CuNPs were applied as antimicrobial agents against eight pathogenic bacteria and five fungi strains. The inhibitory action of the stevia-CuNPs was more pronounced in bacteria than in fungi, and they likewise demonstrated further inhibition zones in Staphylococcus aureus (50.0 mm) than in Aspergillus flavus (55.0 mm). With inhibition zone sizes of 50.0 mm and 47.0 mm and 50 µg/ml minimum inhibitory concentration, S. aureus and A. flavus were the most inhibited pathogens. The minimum lethal effect (MLC) estimate for S. aureus was 50 µg/ml, whereas 75 µg/ml for A. flavus. The stevia-CuNPs mode of action was characterized as bactericidal/fungicidal as the ratio of MIC to MLC was estimated to be equal to or less than 2. After all, stevia-CuNPs could be used as an alternative to commercial antibiotics to solve the problem of multidrug-resistant (MDR) microorganisms.