Background: Acute kidney injury (AKI) is a common complication following cardiac surgery and percutaneous coronary interventions, with an estimated incidence rate around 30%, depicted by long-term intensive care unit stay and culminating renal dysfunction over time, triggering either perpetual renal damage evolving to chronic kidney disease/end-stage renal disease transitions or high vulnerability for sudden death after surgery. The classical diagnosis of AKI is based on a sharp rise in serum creatinine that takes at least 48 h to be visible and is associated with multiple nonrenal factors. Objective: We aimed to evaluate the predictive performance of both neutrophil gelatinase-associated lipocalin (NGAL) and Klotho for AKI in patients who underwent cardiothoracic surgery using cardiopulmonary bypass (CPB). Results: Out of the 182 patients included in the study, 65 had AKI and 117 had non-AKI according to the Kidney Disease: Improving Global Outcomes criteria relying on serum creatinine levels. Baseline serum NGAL was 103.5 ± 41.69 μg/L in the AKI group compared to 79.12 ± 48.02 μg/L in the non-AKI group (p < 0.01) and then manifested a peak-fall-rise pattern until 48 h of CPB, with a more remarkable change in the AKI than in the non-AKI group. ROC curve analysis for all measured biomarkers after 2 h of CPB showed that serum NGAL (0.819, > 75% cutoff, 83.5% accuracy) came after serum creatinine (0.864, > 140% cutoff, 85% accuracy), and troponin I was poorer than both (0.606, > 5.5% cutoff, 60% accuracy). Furthermore, multivariate analysis showed that preoperative serum NGAL, preoperative eGFR ≤60 mL/min/1.73 m2, and arterial hypertension were possible risk factors for AKI with adverse outcomes. Conclusions: Our study suggests the role of preoperative serum NGAL as a prognostic tool for renal consequences after cardiac surgery. Besides, postoperative serum NGAL is a sensitive marker for AKI, but is less specific than serum creatinine. Troponin I is considered to be a risk confirmatory tool and may help in the prediction of AKI. However, its diagnostic utility is restricted due to age-dependent cutoff values and poor standardization and harmonization because of interassay variations.