The invasion of foodborne pathogens remains a serious problem for researchers and regulators in the food industry. The introduction of a new type of potent antibacterial agent is of great importance for controlling bacteria and fungi. This study aimed to investigate a novel antimicrobial agent, such as iron oxide nanoparticles (IONPs), which are biosynthesized by a simple, fast, eco-friendly, and efficient method using Penicillium roqueforti MK805460.1. The obtained iron oxide nanoparticles were covered by SiO2 layer using stöber method. The biosynthesized nanoparticles were characterized by UV-Vis spectroscopy, energy dispersive X-ray (EDX), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Antibacterial activity of the nanoparticles with different concentrations of 50, 100, 150, and 200 μg/ml was examined against Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and bacillus subtilis ATCC 6633) and Gram-negative bacteria (Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 8739) by agar well diffusion and kinetic bacterial growth methods. While, the antifungal activity of nanoparticles was tested against Aspergillus niger, Aspergillus fumigatus, and Aspergillus flavus using the dry weight mycelial method. According to UV-Vis spectroscopic study, the wavelength band observed in the region 204–266 nm, indicating the formation of iron oxide nanoparticles. In addition, spheres of 5–16 nm-sized iron oxide nanoparticles were confirmed via TEM. Also, the results showed that the modified silica nanoparticles had more significant antimicrobial activity than the iron oxide nanoparticles.