Background: Due to their extensive use in medical therapy, iron oxide nanoparticles have recently attracted the attention of researchers in the field of increasing multi-resistance properties in bacterial pathogens. Because iron oxide nanoparticles have a high specific surface area, they can interact with bacterial surface structures and exhibit significant antibacterial activity. Objective: The current work, determined the effect of a novel anti-virulence factor agent which was created from iron oxide nanoparticles against selected gram-positive and gram-negative variant bacterial strains that were isolated and identified from elderly Iraqi patients with urinary tract infections. Methods: Seven bacterial strains (three gram-positive and four gram-negative) were tested for their (biofilm, adhesion, and hemolysis) activity using the quantitative test-tube method, glass-slide method, and by determining the hemolysis ability of bacterial isolates against previously prepared RBC, respectively. The highest virulence factors formation bacterial isolates were chosen to determine the effect of Iron-oxide nanoparticles. Previously prepared and characterized Iron- oxide nanoparticles were used as anti- (biofilm, adhesion, and hemolysis) by using specific Bacterial-Fe3O4 NPs complex with different concentrations. The Results: All these bacteria expressed their virulence factors, the highest-level biofilm formation abilities were detected in Proteus mirabilis and Staphylococcus aureus, and the highest-level adhesion activity was observed in Enterococcus faecalis and Pseudomonas aeruginosa while the highest-level hemolysis activities on human RBC were determined in Micrococcus luteus and E. coli. The effects of (Fe3O4) nanoparticles against the highest virulence factors bacterial isolates shows an increases in the biofilm formation abilities of S. aureus and P. mirabilis as well as for standard bacterial strains the anti-biofilm formation ability of (Fe3 O4) NPs against gram-positive S. aureus, Proteus mirabilis, and standard gram-negative bacteria demonstrated an in-decrease biofilm formation ability of these bacteria effective at (5000 and 10000 µg/ml). The anti-adhesion ability of (Fe3 O4) NPs against gram-positive Enterococcus faecalis, and gram-negative Pseudomonas aeruginosa determined no effect on the adhesion abilities of gram-positive and negative bacteria atall concentrations (250, 400, 500, 750 and 1000µg/ml) Finally, the effect of different concentrations of iron oxide nanoparticles on the hemolysis ability of Micrococcus luteus and Escherichia coli on RBC was determined, the highest hemolysis inhibition level was estimated in 1000 µg/ml and the less inhibition in 500 µg/ml as compared with control.