In view of advantages of green synthesis, a novel green route for synthesis and stabilization of copper nanoparticles (CuNPs) using aqueous extract of Citrus limon fruits at room temperature was reported. The formation of copper nanoparticles (CuNPs) is monitored by recording the UV–vis absorption spectra for surface Plasmon resonance (SPR) peak (∼579 nm). X-ray diffraction (XRD) pattern of the CuNPs agrees with the reported data for Cu metal and the crystallite average size is ~30 nm. Scanning and transmission electron microscopic (SEM and TEM) show uniform spherical particles obtained by this green method. The antimicrobial activity is found to be effective of CuNPs. Results revealed that the green synthesis is an efficient for the preparation of CuNPs as an active antimicrobial agent for practical applications.