Background: Inappropriate prescription of antibiotics is the leading driver of antimicrobial resistance (AMR). The majority of antibiotics are prescribed in primary care. Understanding how general practitioners (GPs) use diagnostic tests and the effect on treatment decision under daily practice conditions is important to reduce inappropriate prescription of antibiotics. The aim of the study was to investigate the use of diagnostic tests in primary care patients with suspected urinary tract infection (UTI) and to assess the appropriateness of the treatment decision (TD) under daily practice conditions in Denmark. Methods: Prospective observational study. Symptomatic adult patients consulting general practice with suspected UTI recruited over 12 months. The diagnostic workup was registered in a standardized form. The appropriateness of the TD was assessed based on the results of a culture performed at a reference microbiological laboratory. TD was considered appropriate if a patient had a positive culture and was prescribed antibiotics or had a negative culture and was not prescribed antibiotics. TD was considered inappropriate if a patient had a negative culture and was prescribed antibiotics (overtreatment) or had a positive culture and was not prescribed antibiotics (undertreatment). Results: Four hundred and eighty-eight patients were included. Dipstick was used in 98% of the patients and urine culture was used in 89% of the patients; 317 had the culture performed in practice and 117 had the culture performed at the hospital. The appropriateness of the final TD was significantly (p = 0.04) lower in patients without culture (55%) than in patients with culture performed in practice (71%) or at hospital (69%). Conclusion: In a context with wide availability of diagnostic tests, GPs use diagnostic tests for the decision-making process in all patients with suspected UTI. Urine culture is used in the majority of the patients and is associated with a higher proportion of appropriate treatment decisions. Performance of urine culture is therefore important in reducing inappropriate antibiotic prescribing in patients with suspected UTI seeking care in general practice in Denmark. Trial registration: ClinicalTrials.gov NCT02249273.