Background: Biofilm is an important problem of great medical concern in which microorganisms are present in extracellular matrix protecting them from external environment, host immunity and antibiotic therapy. Multiple phenotypic methods are present to detect biofilm in vitro tube method, congo red and tissue culture plate methods. objectives: To determine the ability of bacteria that cause urinary tract infection to form biofilm , antibiotic susceptibility pattern in biofilm forming isolates and to detect some of genes responsible for biofilm formation. Methodology: four hundred urine samples were collected 240 samples from catheterized and160 from non catheterized patients who fulfill the inclusion criteria. Samples were cultured and colony forming unit was counted (colony forming unit > 105 were considered positive UTI. Identification of bacteria and their antibiotic sensitivity was done by automated system VITEK II. Multiple phynotypic biofilm detection methods were done and detection of biofilm genes was done by PCR. Results: Enterobacter spp. Were the most frequent isolated organism of Gram negative, Staph aureus was the most frequent isolated organism of Gram positive bacteria. Multiple Phenotypic methods for detection of biofilm production were done to Gram positive and Gram negative bacteria. Tube method detected 84 (68.9%) cases as positive biofilm producer in catheterised patients while 2 (5%) were positive in non catheterized patients. Congo red method detected 80 cases (65.6%) as positive in catheterized cases , 2 (5%) in non catheterized patients but tissue culture plate detected 88 cases(72%) as positive in catheterized patients. Non catheterized patients 18 (45%) were positive. PCR was done to detect biofilm genes ( IcaA, IcaD in staphylococci), (BssS gene in enterobacteriaceae), IcaA, D were detected in 8 (19%) isolates of staphylococci, BssS was detected in 66/104(63.5%) of enterobacteriaceae. Sensitivity of phenotypic methods for biofilm detection in relation to genotypic revealed that tissue culture plate showed more sensitivity in Gram positive and negative bacteria. Conclusion: Multiple phenotypic methods are known for biofilm detection in vitro but tissue culture plate is the most sensitive method; so we can recommend tissue culture plate as a screening method for biofilm detection