BACKGROUND: Urinary tract infections (UTIs) and antibiotic overexposure are common neonatal problems. Recently, evidence has emerged that a standardized approach to neonatal UTI can reduce unnecessary diagnosis without complications. This quality improvement project aimed to achieve those goals in our neonatal intensive care unit (NICU). METHODS: A UTI diagnosis guideline was adapted from the literature with the goal of maximizing the proportion of diagnosed UTIs that conform to accepted diagnostic criteria: >10,000 CFU/mL of one organism with pyuria, or >50,000 CFU/mL of one organism regardless of pyuria. The guideline was implemented in a level III NICU. Adherence, results, and complications were monitored for 12 months. RESULTS: Guideline adherence after implementation was favorable, as evidenced by increased adoption of urinalysis with microscopy. There was a non-significant increase in diagnostic adherence to criteria, 87% to 93%, and non-significant decrease in the rate of UTI diagnosis, 39% to 36%. Complications were not significantly changed. Most UTIs were due to gram-negative rods, especially E. coli; Enterococcus was a common contaminant. CONCLUSIONS: A guideline for diagnosing UTIs can safely increase uniformity. In contrast to previously published results, no significant changes were seen in the rate of UTI or the proportion of diagnoses conforming to the criteria for UTI. It is likely that guideline effects depend on the established practices of NICU providers. Additionally, a consistent pattern was seen in which organisms were more likely to be judged the source of true UTIs or contaminated cultures, meriting further study.