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Background: Early identification of patients at risk for cardiac surgery-associated acute kidney injury (CS-AKI) based on novel biomarkers and tissue oxygen saturation might enable intervention to reduce kidney injury. Aims: The study aimed to ascertain whether brain and muscle oxygenation measured by near-infrared spectroscopy (NIRS), in addition to cystatin C and NGAL concentrations, could help with CS-AKI prediction. Methods: This is a single-centre prospective observational study on adult patients undergoing cardiac surgery using cardiopulmonary bypass (CPB). Brain and muscle NIRS were recorded during surgery. Cystatin C was measured on the first postoperative day, while NGAL directly before and 3 h after surgery. Results: CS-AKI was diagnosed in 18 (16%) of 114 patients. NIRS values recorded 20 min after CPB (with cut-off value ≤ 54.5% for muscle and ≤ 62.5% for the brain) were revealed to be the most accurate predictors of CS-AKI. Preoperative NGAL ≥ 91.5 ng/mL, postoperative NGAL ≥ 140.5 ng/mL, and postoperative cystatin C ≥ 1.23 mg/L were identified as independent and significant CS-AKI predictors. Conclusions: Brain and muscle oxygen saturation 20 min after CPB could be considered early parameters possibly related to CS-AKI risk, especially in patients with increased cystatin C and NGAL levels.