Uropathogenic specific protein is a genotoxic protein targeting the DNA, leading to mutations and modifications in the normal cell's DNA and subsequently, cancer development. This study aims to determine the prevalence of the usp gene in Uropathogenic Escherichia coli isolated from females with urinary tract infections and study its correlation with biofilm formation. One hundred and five urine specimens were collected from female patients (20 to 55 years old) with urinary tract infections attending hospitals. Traditional laboratory methods using selective and differential culture media were used for initial bacterial isolation and identification, and molecular techniques that targeted a segment of the 16SrRNA gene with a specific primer pair were used to confirm the bacterial identification and usp gene detection using a conventional polymerase chain reaction. A microtiter plate method was used to assess the ability of isolates to produce biofilm. The bacterial isolation and identification results revealed (54.28%, 57/105) of isolates were Escherichia coli. The results of molecular detection of the usp gene revealed a considerable prevalence (98.2%, 56\57) in Uropathogenic Escherichia coli and a 100% ability to form a biofilm. The isolates exhibited different biofilm formation abilities, with a higher ability to form strong biofilm (42%, 24/57) followed by moderate and weak biofilm formation (35%,20/57) and (23%, 13/57), respectively. However, no statistical correlation between the usp gene and different abilities for biofilm formation has been found. The study’s limitation is that there is a small number of specimens due to the difficulty in specimen collection. In conclusion, the high prevalence of the usp gene in Uropathogenic Escherichia coli, although it does not correlate with biofilm, suggests its essential role in bacterial pathogenicity and the possibility of cancer disease in females with UTIs.