This study investigated the prevalence of bacterial pathogens of the urinary tract in Australian cats. Urine was collected by cystocentesis and subjected to urinalysis, bacterial culture and susceptibility testing. A total of 126 isolates were obtained from 107 culture-positive cats. Escherichia coli was most commonly isolated (37.3% of isolates) with the majority of isolates showing susceptibility to the 14 antimicrobials tested. Just over a quarter of isolates (27.0%) were Enterococcus faecalis, which showed resistance to cephalosporins and clindamycin. Staphylococcus felis, a previously unreported feline urinary tract pathogen which was susceptible to all antimicrobial agents tested, comprised 19.8% of the isolates. S. felis was significantly associated with urine that had a higher specific gravity (p=0.011) and pH (p=0.006) and was more likely to contain crystals (p=0.002) than urine from which other bacterial species were isolated. This is the first published study that associates the isolation of S. felis with clinical signs of lower urinary tract disease in cats.