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Background A reduction in duration of antibiotic therapy is crucial in minimizing the development of antimicrobial resistance, drug-related side effects and health care costs. The minimal effective duration of antimicrobial therapy for febrile urinary tract infections (fUTI) remains a topic of uncertainty, especially in male patients, those of older age or with comorbidities. Biomarkers have the potential to objectively identify the optimal moment for cessation of therapy. Methods A secondary analysis of a randomized placebo-controlled trial among 35 primary care centers and 7 emergency departments of regional hospitals in the Netherlands. Women and men aged ≥18 years with a diagnosis of fUTI were randomly assigned to receive antibiotic treatment for 7 or 14 days. Patients indicated to receive antimicrobial treatment for more than 14 days were excluded from randomization. The biomarkers procalcitonin (PCT), mid-regional proadrenomedullin (MR-proADM), and C-reactive protein (CRP) were compared in their ability to predict clinical cure or failure through the 10–18 day post-treatment visit. Results Biomarker concentrations were measured in 249 patients, with a clinical cure rate of 94% in the 165 randomized and 88% in the 84 non-randomized patients. PCT, MR-proADM and CRP concentrations did not differ between patients with clinical cure and treatment failure, and did not predict treatment outcome, irrespective of 7 or 14 day treatment duration (ROC AUC 0.521; 0.515; 0.512, respectively). PCT concentrations at presentation were positively correlated with bacteraemia (τ = 0.33, p  < 0.001) and presence of shaking chills (τ = 0.25, p  < 0.001), and MR-proADM levels with length of hospital stay (τ = 0.40, p  < 0.001), bacteraemia (τ = 0.33, p  < 0.001), initial intravenous treatment (τ = 0.22, p  < 0.001) and time to defervescence (τ = 0.21, p  < 0.001). CRP did not display any correlation to relevant clinical parameters. Conclusions Although the biomarkers PCT and MR-proADM were correlated to clinical parameters indicating disease severity, they did not predict treatment outcome in patients with community acquired febrile urinary tract infection who were treated for either 7 or 14 days. CRP had no added value in the management of patients with fUTI. Trial registration The study was registered at ClinicalTrials.gov [ NCT00809913 ; December 16, 2008] and trialregister.nl [ NTR1583 ; December 19, 2008].