Uropathogenic Escherichia coli (UPEC) is prevalent in urinary tract infections (UTIs). UPEC’s biofilm production enables it to invade and persist in the uroepithelium, leading to recurrent UTIs. The biofilm formation is associated with antibiotic resistance. To overcome this resistance, non-conventional compounds must be developed as an alternative to conventional antibiotics. Silver nanoparticles (AgNPs) are significant due to their antibacterial activity against diverse organisms. This study was done to investigate the antibacterial and anti-biofilm effects of AgNPs on UPEC.  AgNPs were biosynthesized using Pseudomonas aeruginosa ATCC 27853. AgNPs were characterized using visual inspection and scanning electron microscopy. The Agar well diffusion method was employed to assess the antibacterial activity of AgNPs against UPEC isolates. The study utilized the tissue culture plate method to investigate both the biofilm and anti-biofilm properties of AgNPs. Following incubation, Ps.aeruginosa and silver nitrate (AgNO₃) mixture exhibited a colour change from pale yellow to dark brown. The mean size of spherical AgNPs observed under a scanning electron microscope was 24.187 ± 8.019 nm. 130 UPECs were obtained. AgNPs exhibited antibacterial activity at a concentration of 20 µg/ml against all tested UPEC strains. Among UPEC strains that produced biofilms, a significant inhibition of 99.89 ± 0.45% was observed at a higher concentration of 512 µg/ml of AgNPs. Ps.aeruginosa produces nitrate reductase enzyme that can potentially convert AgNO₃ to AgNPs. The biosynthesized AgNPs exhibit antibacterial and anti-biofilm activity against all tested UPEC strains.