Several guidelines exist on urinary tract infection (UTI) in children. The objectives of this study were to (1) implement an evidence-based diagnostic guideline on UTI and evaluate determinants of successful implementation, and (2) determine compliance to and impact of the guideline in febrile, non-toilet trained children at the emergency department (ED). We performed a prospective cross-sectional observational study, with observations before and after implementation. Children aged 1 month to 2 years, presenting at the ED with unexplained fever (temperature above 38.5 °C), were included. We excluded children with a chronic underlying disease. Primary outcome measure was compliance to the standardized diagnostic strategy and determinants influencing compliance. Secondary outcome parameters included the following: number of used dipsticks, contaminated cultures, number of genuine UTI, frequency of prescribed antibiotic treatment, and hospitalization. The pre-intervention group {169 children (male 60.4 %, median age 1.0 range 0.1–2.0)} was compared with the post-intervention group {150 children (male 54.7 %, median age 1.0 range 0.1–1.9)}. In 42 patients (24.9 %), there was compliance to local guidelines before implementation, compared with 70 (46.7 %) after implementation ( p -value < 0.001). Improvement in compliance after implementation was higher in patients 3–24 months and outside the office hours ( p  < 0.001). Conclusion : Implementation of a guideline for diagnosing UTI in febrile children at the ED has led to a significantly better compliance, especially in children aged 3–24 months. However, this study also underlines the need for a well-defined implementation strategy after launching an (inter)national guideline, taking determinants influencing implementation into account.