Background: Biofilm is a complex bacterial community that adheres to the surface of implanted biomaterial or mucosa and is a leading cause of numerous chronic infections. These infections are generally difficult to treat and frequently require removal of the biomaterial before the infection can be completely eradicated. The presence of biofilm is associated with increased microbial resistance to antimicrobial therapy and resultant treatment failure. Biofilm formation on indwelling urinary catheters is a leading cause of urinary tract infections (UTI) in hospitalized patients. The continued development of this crystalline biofilm blocks the flow of urine through the catheter triggering episodes of pyelonephritis and septicemia. All available types of indwelling catheters are vulnerable to this problem and currently there are no effective procedures available for its control. The microbiological examination of urine samples from those patients is often misleading, as they can't detect biofilm bacteria colonizing urinary catheters. In view of the large number of infections caused by biofilm producing microorganisms, a reliable method for their diagnosis is important. Aim: Our aim was to find out the incidence of biofilm formation in urinary catheterized patients and to detect a reliable diagnostic technique for the detection of the formed biofilm. Materials and methods: The study was done on 20 hospitalized patients, half from pediatrics surgery ward and the other half from medical wards (Cairo University Hospitals), with indwelling catheters for ≥ 3 days.The age of the patients ranged from 1.5 to 85 with a mean age of 34.75 ± 3.11 [mean ± standard deviation (SD)]. Catheters remained in situ for a period of 3 to 20 days (mean 8 ±5.22 days.The samples were catheter segments and urine. Most of the patients 18/20 (90%) were on antimicrobial therapy in the forms of 2nd and 3rd generation cephalosporins, aminoglycosides, amoxycilline and flucloxacillin, ampicillin/salbactam and vancomycin. All the 20 samples were cultured on CLED agar and the microorganisms isolated from the catheter and urine samples were identified by the standard microbiological methods. Segments of the 20 catheter samples were examined by the scanning electron microscopy (SEM) for the presence of biofilm. The organisms isolated from catheter cultures were tested for their ability to produce biofilm in vitro. Using the tissue culture plate (TCP) which is semi-quantitative determination of biofilm formation in 96-well flat bottom plates. The adherence of the tested strains isolated from catheter cultures to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The measurements also agreed with visual assessments of bacterial adherence to culture tubes, and tissue culture plates. Results: Out of the 20 urine samples cultured, 9/20 (45%) showed positive culture and the microbial species isolated were; E.coli, Acinetobacters, C. albicans, non C. albicans. Catheter cultures showed 12/20 (60%) positive culture results with isolated microbial species similar to those isolated from urine samples. There was no correlation between patients having renal conditions and biofilm formation (p=0.336) and no significant correlation was found between the duration of catheter in situ and biofilm formation (p=0.095). There are associated conditions (e.g. DM, renal insufficiency etc.) that enhance the development of UTI and biofilm but we neither found a significant correlation between the associated conditions and UTI nor a significant one between the same conditions and biofilm formation (p =0.836, 0.163 respectively).There was reduction in microbial diversity with antimicrobial use but the correlation was insignificant (p=0.317). SEM images of 14/20 (70%) showed biofilms on the luminal surface and the remaining 6/20 (30%) showed no biofilm. The results of urine and catheter cultures correlated well with the results of the SEM (p values, 0.008 and 0.000 respectively). Lastly, 4/12 (33.33%) of the isolates from catheter samples (which showed biofilm by SEM) were biofilm producers by TCP assay. The correlation between urine culture result and the result of TCP assay was insignificant (p =0.237).The sensitivity, specificity, total accuracy, PPV and NPV of urine culture and catheter culture against SEM were, 64.30%, 100%, 75%, 100%, 54% and 85.70%, 100%, 90%, 100%, 75% respectively. Unfortunately the sensitivity, specificity, total accuracy, PPV and NPV of TCP assay could not be calculated because the results of the TCP assay were all positive, no negative results. Conclusion: The use of indwelling urinary catheters remains widespread and is associated with chronic infections due to the formation of biofilms on the catheter surface.Microbiological analysis of the intraluminal surface of the catheter is required for early identification of the causative agents of catheter associated urinary tract infections (CAUTI) and biofilms on urinary catheters combined with a routine microbiological examination of urine. SEM proved to be a reliable method for the detection of biofilm on urinary catheters than urine culture and TCP assay.