BACKGROUND The aim of this study was to evaluate antibiotic resistance and biofilm formation of Staphylococcus aureus strains, isolated from urine samples of patients with urinary tract infection. The samples were collected in the period of 2005-2009 and originated from patients of St. Anne's Teaching Hospital in Brno. METHODS A total of 128 S. aureus strains from 128 patients with the diagnosis of urinary tract infection were examined. In addition to testing the antibiotic resistance to doxycycline, vankomycin, nitrofurantoin, oxacillin, amoxicillin/clavulanate, cefoxitin and sulfamethoxazol/trimethoprim, biofilm was also detected in 112 strains by the modified Christensen method. RESULTS No S. aureus strain resistant to vankomycin and nitrofrantoin was found, 12 strains were resistant to oxacillin (9.4 %), 6 to doxycyklin (4.7 %), 9 to amoxicillin/clavulanate (7.0 %) as well as to cefoxitine, and 2 to sulfamethoxazol/trimethoprim (1.6 %). Out of 20,375 positive urine samples examined in our laboratory between 2005 and 2009, S. aureus was detected as a causative agent of urinary tract infection in 0.6 %, being more frequent in males. Biofilm was produced by 14 (12.5 %) out of 112 S. aureus strains. CONCLUSIONS The biofilm formation of urinary S. aureus strains was low. The strains showed higher resistance to beta-lactams, in other cases resistance was low. S. aureus was found to be more frequent in urine of older patients, males and after previous interventions using instrumental.