We report an eco-friendly method for the synthesis of copper nanoparticles (CuNPs) using Citron juice (Citrus medica Linn.), which is nontoxic and cheap. The biogenic copper nanoparticles were characterized by UV-Vis spectrophotometer showing a typical resonance (SPR) at about 631 nm which is specific for CuNPs. Nanoparticles tracking analysis by NanoSight-LM20 showed the particles in the range of 10-60 nm with the concentration of 2.18 × 10(8) particles per ml. X-ray diffraction revealed the FCC nature of nanoparticles with an average size of 20 nm. The antimicrobial activity of CuNPs was determined by Kirby-Bauer disk diffusion method against some selected species of bacteria and plant pathogenic fungi. It was reported that the synthesized CuNPs demonstrated a significant inhibitory activity against Escherichia coli followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes and Salmonella typhi. Among the plant pathogenic fungi tested, Fusarium culmorum was found to be most sensitive followed by F. oxysporum and F. graminearum. The novelty of this work is that for the first time citron juice was used for the synthesis of CuNPs.