OBJECTIVE : The synthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities was investigated and extracellularly biosynthesized using Penicillium italicum isolated from Iraqi lemon fruits. RESULTS : The formation of synthesized nanoparticles was observed after 72 h of incubation. Color changing, ultraviolet and visible spectrum, X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy and energy dispersive analysis X- ray confirmed the biosynthesis and characterization of silver nanoparticles. Ultraviolet and visible spectroscopy showed silver surface plasmon resonance band at 415 nm. X- ray diffraction showed that the particles were crystalline with face-centered cubic lattice phase and a size of 39.5 nm. Fourier transform infrared analysis shows the possible interactions between silver and bioactive molecules, which may be responsible for synthesis and stabilization of silver nanoparticles. Scanning electron microscopy imaging revealed different morphologies of the AgNPs, some of nanoparticles were close to spherical in shape, while others had platelet like structure, in size range between 32 and 100 nm. The transmission electron microscopy image demonstrated that AgNPs were spherical with a size < 50 nm. The biosynthesized silver nanoparticles were proved to be potential antioxidants by showing effective radical scavenging activity against 2, 2-diphenyl-1-picrylhydrazyl, hydroxyl radical and resazurin. The nanoparticles also showed potent antimicrobial activity against various pathogens, including bacteria and fungi as well as significant concentration-dependent cytotoxic effects against human breast cancer cells. CONCLUSIONS : The powerful bioactivity of the synthesized silver nanoparticles recommends their biomedical use as antioxidant, antimicrobial as well as cytotoxic agents.