A urinary tract infection (UTI) occurs when bacteria enter and multiply in the urinary system. The infection is most often caused by enteric bacteria that normally live in the gut, which includeEnterococcus faecium. Without antibiotic treatment, UTIs can progress to life-threatening septic shock. Early diagnosis and identification of the pathogen will reduce antibiotic use and improve patient outcomes. In this work, we develop and optimize a cost-effective and rapid (< 40 min) method for detectingE. faeciumin urine. The method uses a fluorescently labelled bacteriocin enterocin K1 (FITC-EntK1) that binds specifically toE. faeciumand is then detected using a conventional flow cytometer. Using this detection assay, urine containingE. faeciumwas identified by an increase in the fluorescent signals by 25–73-fold (median fluorescence intensity) compared to control samples containingEscherichia coliorStaphylococcus aureus. The method presented in this work is a proof of concept showing the potential of bacteriocins to act as specific probes for the detection of specific bacteria, such as pathogens, in biological samples.