The synthesis of nickel oxide nanoparticles in film form using dead biomass of the filamentous fungus Aspergillus aculeatus as reducing agent represents an environmentally friendly nanotechnological innovation. The optimal conditions and the capacity of dead biomass to uptake and produce nanoparticles were evaluated by analyzing the biosorption of nickel by the fungus. The structural characteristics of the film-forming nickel oxide nanoparticles were analyzed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). These techniques showed that the nickel oxide nanoparticles had a size of about 5.89 nm and were involved in a protein matrix which probably permitted their organization in film form. The production and uptake of nickel oxide nanoparticles organized in film form by dead fungal biomass bring us closer to sustainable strategies for the biosynthesis of metal oxide nanoparticles.