Background: Surgical cryothermia and radiofrequency (RF) ablations for atrial fibrillation (AF) seem to result in similar sinus rhythm restoration, but the biochemical consequences of the two methods are unclear. We aimed to compare the biochemical responses to the two ablative methods in concomitant mitral valve surgery (MVS). Methods: Sixty mitral valve surgery patients with AF were prospectively included. Forty-one patients planned for ablation were randomized to cryothermia (n = 20) or radiofrequency (n = 21) ablation and 19 served as controls. Markers for myocardial injury, inflammation, cell stress, apoptosis, and heart failure were analyzed pre-and postoperatively at different time points. Results: Troponin T and creatine kinase isoenzyme MB (CK-MB) peak levels were significantly higher in the cryothermia group compared with the RF group (12,805 [6140-15,700] vs. 2790 [1880-4180] ng/L; P = 0.002 and 271 [217-357] vs. 79 [66-93] μg/L; P < 0.001, respectively). Both groups had significantly higher levels than the noablation group. There were no group differences in C-reactive protein (CRP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), but there were correlations between pre-and postoperative levels of both CRP (r s = 0.41, P = 0.001) and NT-proBNP (r s = 0.48, P < 0.001). Protease-activated receptor 1 (PAR-1) and heat shock protein 27 (HSP27) were significantly increased in the cryoablation group. Conclusions: Cryoablation results in a larger myocardial injury and possibly more elevated apoptotic activity and cell stress compared with the RF technique. The type of ablation device did not have any significant influence on the postoperative inflammatory response nor on the early postoperative levels of NT-proBNP.