Abstract Background Urinary tract infection (UTI) is one of the most common diagnoses in girls and women, and to a lesser extent in boys and men younger than 50 years. Escherichia coli, followed by Klebsiella spp. and Proteus spp., cause 75-90% of all infections. Infection of the urinary tract is identified by growth of a significant number of a single species in the urine, in the presence of symptoms. Urinary culture is an accurate diagnostic method but takes several hours or days to be carried out. Metabolomics analysis aims to identify biomarkers that are capable of speeding up diagnosis. Methods Urine samples from 51 patients with a prior diagnosis of Escherichia coli-associated UTI, from 21 patients with UTI caused by other pathogens (bacteria and fungi), and from 61 healthy controls were analyzed. The 1H-NMR spectra were acquired and processed. Multivariate statistical models were applied and their performance was validated using permutation test and ROC curve. Results Orthogonal Partial Least Squares-discriminant Analysis (OPLS-DA) showed good separation (R2Y = 0.76, Q2=0.45, p