ObjectivesTo determine the performance of procalcitonin (PCT), C reactive protein (CRP) and absolute neutrophil count (ANC) in identifying invasive bacterial infection (IBI) among well-appearing infants ≤21 days old with fever without source and no leukocyturia. To compare this performance with that in those 22–90 days old.DesignSubstudy of a prospective single-centre registry performed between September 2008 and August 2017.SettingPaediatric emergency department of a tertiary teaching hospital.Patients196 infants ≤21 days old and 1331 infants 22–90 days old.Main outcome measuresSensitivity and negative likelihood ratio of blood tests for ruling out IBI (positive blood or cerebrospinal fluid culture). Abnormal blood test results: PCT ≥0.5 ng/mL, CRP >20 mg/L and ANC >10 000/µL.ResultsPrevalence of IBI in infants ≤21 days old with normal or any abnormal blood test result was 3.6% and 6.8%, respectively (OR 0.52 (95% CI 0.13 to 2.01)), compared with 0.2% and 4.5% in older infants (OR 0.03 (95% CI 0 to 0.17)). Sensitivity and negative likelihood ratio of the blood tests for ruling out IBI in infants ≤21 days were 44.4% (95% CI 18.9% to 73.3%) and 0.79 (95% CI 0.43 to 1.44), respectively (vs 84.6% (95% CI 57.8% to 95.7%)%) and 0.19 (95% CI 0.05 to 0.67) in older infants). The values improved in infants with fever ≥6 hours aged 22–90 days, but not in those ≤21 days.ConclusionsPCT, CRP and ANC are not useful for ruling out IBI in febrile infants ≤21 days old. It is still recommended that these patients are admitted and given empirical antibiotic therapy, regardless of their general appearance or blood test results.