Background: Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common complication of cardiac surgery and is associated with increased morbidity and mortality. Recent guidelines emphasize the need for new monitoring methods to facilitate targeted CSA-AKI prevention and treatment strategies. In vivo real-time measurement of mitochondrial oxygen tension (mitoPO(2)), could potentially fulfil this role during cardiac surgery, as suggested in our previous pilot study. Methods: In this prospective observational study, we investigated 75 cardiac surgery patients with an increased preoperative CSA-AKI risk. The primary aim of this study was to assess whether patients who developed CSA-AKI experienced prolonged periods of mitoPO(2) < 20 mmHg during surgery. mitoPO(2) was measured intraoperatively, and CSA-AKI was defined according to the Kidney Disease: Improving Global Outcomes criteria. Four additional mitoPO(2) thresholds (<25, <30, <35, and < 40 mmHg) were analyzed, including the predictive capacity of all thresholds for CSA-AKI. Results: This study found that patients who developed CSA-AKI had a significantly longer intraoperative time with mitoPO(2) <20 mmHg and <25, <30, <35, and <40 mmHg. Subsequently, we tested all thresholds for their association with the risk of CSA-AKI, with the <25 mmHg threshold demonstrating the highest significant odds ratio. Every minute spent below <25 mmHg increased the risk of CSA-AKI by 0.7 % (P = 0.021). Conclusions: This study highlighted the association between mitoPO(2) and the onset of CSA-AKI. Extended durations below the mitoPO(2) threshold of 25 mmHg significantly correlate with an elevated CSA-AKI risk. Using mitoPO(2) as a monitoring tool shows promise in potentially predicting and possibly preventing CSA-AKI when used as a treatment trigger in cardiac surgery patients.