© 2019 Mexican Journal of Biotechnology. All rights reserved. The ability of four extracellular filtrates (FE), obtained from A. alternata biomass grown in four different culture media; malt dextrose broth (MDB), potato dextrose broth (PDB), sucrose (S) and Czapek (C), were evaluated to biosynthesize iron nanoparticles (FeNPs), at the end of the assay a brown color was revealed in all the samples. To evidence biosynthesis of nanoparticles, absorption spectra were made from 200 to 600 nm for all the samples and, analyzed by transmission electronic microscopy (TEM). The morphology and size of the synthesized FeNPs were studied using the SPIP 6.2.0 software. From the four extracellular filtrates assays, the nanoparticles biosynthesis was possible only in the EF-PDB and EF-C, showing their presence in the UV-vis spectrum when forming an absorption band at 226 and 225 respectively, and plateau around at 270 nm to EF-PDB, and 290 nm to EF-C. The synthesized nanoparticles presented spherical and polydisperse form, those synthesized with EF-PDB showed a size of 20-40 nm, while those synthesized with EF-C had a size of 10-80 nm. Six months after their production, the FeNPs biosynthesized by EF-C were analyzed by microscopy showing an increase forming 5 µm microparticles, possibly due to magnetic attraction of these materials. To EF-S a peak at 231 nm was detected, but TEM images show formation of iron microcrystals, and iron crystals in EF-MDB which maximum intensity of absorption between 225-303 nm. Culture media used to produce the fungus biomass and get EF have influence in the iron nanoparticles biosynthesis, even in the case of the same microorganism. The FeNPs biosynthesized by A. alternata could be used in the removal of contaminants or as microbial agents against bacteria of clinical or agricultural importance.