The aim of study was to determine the effects of ozone therapy on the oxidative stress, cardiac functions and clinical findings in patients with heart failure reduced ejection fraction (HFrEF). A total of 40 patients with New York Heart Association 2 and 3 HF with left ventricular ejection fraction (LVEF) < 35%, and 40 subjects without HF as control group were included in the study. Patients with HFrEF were given additional ozone therapy of major and minor administrations along with conventional HF treatment for 5 weeks. Before and after ozone therapy, left ventricular end-systolic and end-diastolic volumes (LVESV, LVEDV) and the 6 minute walk distance (6MWD) and blood levels of the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSHPx), malondialdehyde (MDA), nitric oxide (NO) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured. Ozone therapy significantly reduced the serum levels of NO and MDA (p < 0.001, respectively) and significantly increased the levels of SOD, CAT, GSH and GSHPx (p < 0.001, respectively). LVEDV and LVESV were found to be significantly reduced; however, LVEF was not found to be significantly increased (p = 0.567). As the biochemical improvement marker of HF, NT-proBNP was significantly reduced (p < 0.001). The clinical HF improvement marker of 6 minute walk distance was also modestly increased (p < 0.001). Ozone therapy might be beneficial in terms of activating antioxidant system and merit further therapeutic potential to conventional HF treatment in patients with HFrEF.