THE PATHOGENICITY of the bacteria is significantly influenced by the virulence factors, such as biofilm. Urinary tract infections (UTIs), a common urologic condition that affects millions of individuals worldwide, are caused by bacteria that are extremely resistant to antimicrobials. Therefore, the goal of this work was to use nanoparticles as contemporary antimicrobials to discover a solution to this worldwide issue. For the first time, Enterococcus faecalis isolates from food sources were used to naturally create iron oxide nanoparticles (E-IONPs). The biosynthesis process was done in optimized conditions at 1M of concentration of iron oxide solution at 60 ˚C for the incubation period of 24h at pH 5. The bacterial isolates were isolated from clinical and food samples. After being identified, food-origin bacteria were used in the biosynthesis process, while the clinical isolates were evaluated for their ability to form biofilm. E-IONPs were firstly characterized by color change, then by UV-vis spectroscopy, AFM, FTIR, and SEM analysis. The anti-biofilm activity of the super-magnetic E-IONPs was assessed using a microtiter plate assay. Our results revealed that the biosynthesized E-IONPs were cubic and irregular in shape, and they have an anti-biofilm activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, and Streptococcus agalactiae. Eventually, the biosynthesized super-magnetic iron oxide nanoparticles were concluded to be effective against biofilm formed by uropathogenic bacteria.