Recently, biological techniques for manufacturing nanoparticles, such as employing filamentous fungi to synthesize ZnO nanoparticles, have become environmentally friendly, bio congruous, and safe. This study aimed to look for Penicillium italicum [Filamentous Blue Mold (FiBM)] in rotting citrus fruits and exploit this in the biofabrication of ZnO nanoparticles. The study isolated 39 different filamentous mold samples and used conventional and molecular diagnosis. Only 11 (28%) of the isolates obtained contained Penicillium italicum, for which we investigated the capability of ZnO nanoparticles biosynthesis by fungal extracellular free-cells filtrate solution. The results showed that Penicillium italicum Pit-L6 was given the peak of ZnONps 378 nm detected by UV-visible spectrophotometry, and it considered significantly optimum strain in the highest quantity (mean±S.D) 0.015±0.002 gm/100 ml with small enough average nanoparticles size. The ZnONps were characterized by UV-visible scanning spectrophotometry, atomic force microscopy (AFM), X|-RD, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The final average ZnONps through 0f in all measuring devices ranged between 53.13–69.67 nm (with different shapes and dimensions). This study concluded that these fungi (FiBMs) are highly capable as eco-friendly and cheap bio-nano factories to manufacture ZnONps as alternative novel biological technology, in fine particles within average size at nano-level, as continuous renewable sources for producing nanoparticles, for different usage.