One of the most significant pathogens involved in urinary tract infections is Uropathogenic E. coli (UPEC). In addition to assessing biofilm production and the effect of gold and silver nanoparticles on the isolated strains’ ability to form biofilms, the study intended to determine the prevalence of multidrug-resistant (MDR) UPEC in patients who were admitted to Menoufia University Hospitals with both hospital-acquired (HA) and community-acquired (CA) urinary tract infections (UTIs). E. coli strains were isolated from 312 urine samples, and the antibiotic resistance profile of the isolated strains was evaluated using the Kirby-Bauer disc diffusion technique. Using the tissue culture plate method, biofilm production was identified, and certain virulence genes were found. Lastly, biofilm development following incubation with different concentrations of both nanoparticles was measured to assess how well gold and silver nanoparticles inhibited biofilm formation. From 312 urine samples used, 100 E. coli were isolated. Of these isolates, 58 (58%) were isolated from HA-UTI and 42 (42%) from CA-UTI patients. Biofilm was produced by 89.5% of catheterized and 80% of non-catheterized HA E. coli, compared to 66.7% of CA isolates. MDR rates were not 44.7% for catheterized, 45% for non-catheterized hospital-acquired and 33.3% for community-acquired E. coli isolates. About 96% produced FimH, 24% produced Sfa and 68% produced IutA. Antibofilm effect of silver was much better than gold nanoparticles. FimH, Sfa and IutA were more predominant among HA isolates than community. Biofilm formation is effectively inhibited by silver nanoparticles (AgNPs). Therefore, AgNPs can be used in medical devices to stop biofilms from forming, whereas gold had a much less effective antibiofilm effect.