Escherichia coli is the dominant bacterial cause of UTI among the uropathogens in both developed and developing countries. This study is to investigate the effect of Acacia nilotica aqueous extract on the survival and biofilm of isolated pathogens to reduce UTIs diseases. A total of 170 urine samples were collected from Luxor general hospital and private medical analysis laboratories in Luxor providence, Egypt. Samples were screened for the incidence of uropathogens by biochemical tests, antibiotics susceptibility, detection of virulence, and antibiotic-resistant genes by multiplex PCR, biofilm formation, and time-killing assay. Escherichia coli is by far the most prevalent causative agent with the percentage of 73.7% followed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeuroginosa, and Acinetobacter baumanii. Isolates were multidrug-resistant containing blaTEM, blaSHV, blaCTX, qnrs, and aac(3)-Ia resistant genes. All isolates were sensitive to 15-16.7 mg ml-1 of Acacia nilotica aqueous extract. Time killing assay confirmed the bactericidal effect of the extract over time (20-24 h). A high percentage of 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl (23.5%); á-Selinene (15.12%); Oleic Acid (14.52%); Globulol (11.35%) were detected among 19 bioactive phytochemical compounds in the aqueous extract of A. nilotica over the GC-mass spectra analysis. The plant extract reduced significantly the biofilm activity of E. coli, K. pneumoniae, P. mirabilis, and P. aeuroginosa by 62.6, 59. 03, 48.9 and 39.2%, respectively. The challenge to improve the production of A. nilotica phytochemicals is considered a very low price for the return.