The current diagnostic standard procedure outlined by the Health Protection Agency for urinary tract infections (UTIs) in clinical laboratories does not report bacteria isolated from samples containing three or more different bacterial species. As a result many UTIs go unreported and untreated, particularly in elderly patients, where polymicrobial UTI samples are especially prevalent. This study reports the presence of the major uropathogenic species in mixed culture urine samples from elderly patients, and of resistance to front-line antibiotics, with potentially increased levels of resistance to ciprofloxacin and trimethoprim. Most importantly, the study highlights that Escherichia coli present in polymicrobial UTI samples are statistically more invasive (P<0.001) in in vitro epithelial cell infection assays than those isolated from monomicrobial culture samples. In summary, the results of this study suggest that the current diagnostic standard procedure for polymicrobial UTI samples needs to be reassessed, and that E. coli present in polymicrobial UTI samples may pose an increased risk to human health.