Human and animal UTIs have increasingly become prevalent posing severe health risks to everyone infected. The purpose of this work was to identify bacterial UTIs in humans and cows, as well as to investigate antibiotic resistance of Klebsiella pneumoniae isolates concerning biofilm production. A total of 100 urine samples were collected: 50 from humans admitted to Al-Hashimiya and Al-Qasim General Hospital, Iraq, and 50 from cows in different areas of Babylon province in September- December 2021. Three genes were targeted: WCAg, TRE-C, and the housekeeping gene rrsE. Among 100 cases, 45 bacterial isolates were identified: K. pneumoniae (22 isolates, 48.88%), Escherichia coli (10 isolates, 22.22%), Staphylococcus aureus (7 isolates, 15.55%), and Pseudomonas aeruginosa (6 isolates, 13.33%) Klebsiella pneumoniae biofilm formation was determined using congo red agar and microtiter plate method, detecting that 2 exhibited strong biofilm forming ability, 8 moderate ability, 10 weak ability and 2 no biofilm forming ability. Relative expressions of wcaG, treC, and rrsE were validated by qPCR. AST indicated moderate susceptibility of the isolates to piperacillin-tazobactam (54.54%), cefepime (50%), cefotaxime (50%), cefoperazone-tazobactam (45.45%), ceftazidime (45.45%), and amoxicillin-clavulanic acid (40.4%). That is why in anamnestic data the sensitivity of other antibiotics such as amoxicillin, cefuroxime, cefoxitin, and ceftriaxone was below 40%. This increase has been associated with multidrug-resistant K. pneumoniae, through presented mutations stimulating biofilm formation – a factor difficult to treat.