The most common cause of urinary tract infections (UTI) is uropathogenic Escherichia coli, which is often resistant to antibiotics. E. coli can form biofilms on urinary catheters. The biofilm protects E. coli against various factors. In this study, silver nanoparticles (AgNPS) were synthesized using Trifolium pratense L. extract as a reducing agent. The AgNPs were characterized by visible UV spectroscopy, a diffraction pattern (XRD), transmission electron microscopy (SEM), and energy dispersion spectroscopy (EDX). The AgNPs had a spherical shape with an average particle of 19 nm. Biological properties were also evaluated using biofilm inhibition, anticancer, antimicrobial activity. The brine shrimp lethality assay was applied to evaluate the anticancer activity of the nanoparticles. The silver nanoparticles with LC50 (1.3 μg/ml) had the highest cytotoxicity activity. The antimicrobial activity of nanoparticles was evaluated by the agar diffusion method, minimum inhibitory concentration, and minimum bactericidal concentration in the range of 1.00 to 0.0312 and 2.00 to 0.0312, respectively. The nanoparticles exhibited a high antimicrobial effect against human E.coli pathogenic strains isolated from Urinary Tract Infection. The effect of biofilm inhibition on antibiotic resistant clinical strains by silver nanoparticles showed that silver nanoparticles inhibited biofilm between 21.12 to 97.10%.