Nanotechnology is one of the most emerging fields in the recent years. In the current investigation, we report the biosynthesis of iron nanoparticles (Fe-NPs) employing Alternaria alternata fungus, which is an eco-friendly process for the synthesis of metallic nanoparticles. Fe-NPs were synthesized through the reduction of aqueous Fe3+ ion in the dark conditions. Ultraviolet–visible spectrum of the aqueous medium containing iron ion showed a peak at 238 nm and another peak at 265 nm. The forming of nanoparticles was confirmed by transmission electron microscope, scanning electron microscope and energy-dispersive x-ray. The morphology of nanoparticles is found to be cubic shapes mostly and the average particle diameter as determined by scanning electron microscope was found to be 9±3 nm. Fe-NPs showed antibacterial activity against both Gram-positive and Gram-negative bacteria used in this study due to its oxidative damage for bacterial cell wall. Iron nanoparticles show more antimicrobial activity to Bacillus subtilis than Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Key words: Mycosynthesis, Alternaria alternata, iron nanoparticles, antibacterial.