Antibiotic resistance driven by antibiotic use remains a major public health and professional concern. Our aim was to know the local prevalence of uropathogens and their antimicrobial susceptibility profile in acute pyelonephritis. A prospective study of patients admitted in a level III Pediatric Department ward with acute pyelonephritis from 1994 to 2012 was performed in Northern Portugal. Etiological agents and their antimicrobial sensitivity profile were evaluated in four timed periods (G1: 1994-97; G2: 2002; G3: 2007; G4: 2012). We evaluated 581 patients, 66% female with median age 22 months. Escherichia coli was the leading uropathogen and its prevalence remained stable during the last 18 years. It showed an increased sensitivity to amoxicillin-clavulanate from 71% in G1 to 81.5% in G4 (p = 0.001) and a decreased resistance rate from 8.7% in G1 to 2.8% in G4 (p = 0.008). Its sensitivity to 2nd and 3rd generation cephalosporin was more than 90% (p = ns) and more than 95% to nitrofurantoin (p = ns). Resistance rate of cotrimoxazole increased from 22% to 26% (p = 0.008). Escherichia coli remains the main uropathogen responsible for acute pyelonephritis, reason why its antimicrobial sensitivity profile will determine the empirical therapeutic choice. Amoxicillin-clavulanate remains a good first-line choice for empirical treatment of acute pyelonephritis in our inpatient health care.