OBJECTIVE To characterize uropathogenic Escherichia coli (UPEC) in cases of clinical feline urinary tract infection (UTI) and subclinical bacteriuria and investigate the in vitro effects of E coli strain Nissle 1917 on isolate growth. ANIMALS 40 cats with positive E coli culture results for urine collected during routine evaluation. PROCEDURES Characterization of UPEC isolates was performed by PCR-based phylotype analysis and serotyping. Nissle 1917 effects on growth inhibition and competitive overgrowth against UPEC isolates were evaluated in vitro using a plate-based competition assay. RESULTS Feline phylogroups were similar to previous human and feline UPEC studies, with most of the isolates belonging to phylogroup A (42.5%), B2 (37.5%), and D (15.0%). Fifty-two percent of isolates were found to be resistant to antimicrobials, with 19% of these being multidrug resistant (MDR). Nissle 1917 adversely affected the growth of 82.5% of all isolates and 100% of MDR isolates in vitro. The median zone of inhibition was 3.33 mm (range, 1.67 to 10.67 mm). Thirteen isolates were affected via competitive overgrowth and 20 via growth inhibition. CLINICAL RELEVANCE UPEC isolates from cats were similar in phylogroup analysis to human and dog isolates. The in vitro effects of Nissle 1917 on UPEC warrant additional studies to determine if similar results can be duplicated in vivo.