Background Urinary tract infection (UTI) diagnosis in infants is often made by a positive urine culture result, regardless of urine dipstick findings. Aim To assess parameters that may affect positive urine culture results interpretation in infants, including dipstick performance, obtainment method, bacteria type, age and laboratory results. Methods A retrospective, cohort study. Infants <90 days with urine dipstick and culture obtained through subrapubic aspiration (SPA) or catheter, 2015-2016, were included. Results Overall, 19% (129/678) of cultures were positive. The dipstick sensitivity was 51% for all cultures; 66%, 47%, 15% and 10% for Escherichia coli (n= 71), Klebsiella (n= 19), Enterococcus (n= 34) and Proteus (n= 10), respectively (p<.001). Sensitivity was higher in SPA vs. catheter for all cultures (67% vs. 43%); E. coli (78% vs. 59%); and Klebsiella (88% vs. 18%). For Enterococcus, dipstick sensitivity was low in both SPA and catheter (0-16%). All Proteus episodes were catheter obtained. Positive culture with negative dipstick and Enterococcus episodes had lower C-reactive protein levels, and higher proportion of mixed infection compared with positive dipstick and E. coli episodes. Conclusions Urine cultures in infants should be obtained by SPA, since catheter-obtained, Enterococcus and Proteus positive cultures may represent contamination or asymptomatic bacteriuria, rather than true UTI.