Haemolysis occurring during cardiac surgery with cardiopulmonary bypass (CPB) is assumed to be a risk factor for postoperative acute kidney injury (AKI). Plasma alpha-1 microglobulin (A1M) may have a protective role as haem scavenger. The aim of this study was to evaluate the association between AKI and the degree of haemolysis and the course of A1M concentrations during cardiac surgery, respectively. We analysed plasma concentrations of free haemoglobin (pfHb) and A1M in 25 patients undergoing cardiac surgery: before CPB; during CPB in 15 min intervals; after CPB; and at four additional time points until 24 h after surgery. Markers of kidney function were followed until 4 days after surgery. Detection of AKI was based on the KDIGO (Kidney Disease, Improving Global Outcome) criteria. The plasma concentration of free haemoglobin during CPB was found to be significantly higher in patients with postoperative AKI at 60 min after start of CPB [mean 1379 µg/mL (95% CI: 1037–1721)]; compared to [820 µg/mL (622–1018)]; p = 0.034, in patients without AKI, and at one hour post-CPB [2600 µg/mL (969–4230)] vs [1037 µg/mL (722–1353)]; p = 0.044]. There was no significant difference found for pA1M levels between the groups with and without postoperative AKI development. Haemolysis during cardiac surgery with CPB increases the risk of postoperative AKI. Levels of pA1M did not differ for patients who developed postoperative AKI compared with those who did not. The data did not allow conclusions regarding the hypothesis that pA1M has a reno-protective effect.