Heart failure (HF) is a condition associated with the apoptosis and cell death of both cardiac myocytes and cardiac non-myocytes. DNA fragments released from programmed cell death or acute cellular injury are the main sources of disease-associated elevation of cell-free (cf) DNA. We hypothesized that cfDNA could be a relevant marker of cardiac apoptosis in HF patients that could be affected by the improvement of myocardial performance. To test our hypothesis, we measured plasma cfDNA in 19 patients with ischemic HF and severe left ventricular (LV) systolic dysfunction before and 12 h after completion of levosimendan infusion. Echocardiographic and biochemical markers of LV diastolic pressure and LV systolic function were also assessed. In accordance with previous observations levosimendan improved echocardiographic and biochemical indices of LV function. Plasma cfDNA was significantly reduced in HF patients post-levosimendan treatment (median: 89.4, interquartile range: 87.1 to median: 25.9, interquartile range: 12.3, P = 0.028). Notably, in 15/19 patients there was a reduction in cfDNA levels post-levosimendan infusion; while in 12/19 patients, a more than 50% reduction in plasma cfDNA was observed. Since cfDNA is a marker of tissue injury and apoptosis these results indicate that improvement of LV function has a potential impact on cell preservation and survival. Further studies are needed to substantiate our promising results regarding the role of plasma cfDNA as a marker of HF.