Background The aim of this study was to compare the estimated glomerular filtration rate (eGFR) from serum creatinine (eGFRcrea) and cystatin C (eGFRcys) in patients with elevated serum trough levels of gentamicin before the next planned dose during treatment in the intensive care unit (ICU). Methods This was a retrospective observational study. Patients who stayed in an ICU, received a once-daily dose of gentamicin, and who had results from all serum gentamicin trough levels, eGFRcrea and eGFRcys analyses were included in the study. Overdosed patients were defined as those with serum gentamicin trough levels above 1 mg/L before the next dose. Gentamicin was measured by a particle-enhanced turbidimetric immunoassay (PETIA). Creatinine and cystatin C were measured by standardized methods. Results The median age (range) was lower in all patients with gentamicin concentration measurements than in overdosed patients (67 [19–96] vs. 75 [48–99] years, respectively; p 2 vs. 41 [29–58] mL/min/1.73 m2, respectively; p 2 vs. 66 [54–93] mL/min/1.73 m2, respectively; p Conclusions Overdosed patients had both a lower eGFRcrea and eGFRcys than controls. Elderly patients are the most commonly overdosed patients. We recommend measuring cystatin C and calculating the eGFRcys and combined equation (eGFRcrea + cys) in ICU patients over 65 years of age, which would enable improved gentamicin dosage adjustments.