Sepsis is associated with the activation of white blood cells (WBCs) that leads to the production of extracellular traps. This process increases extracellular DNA (ecDNA) that can be recognized by the innate immune system and leads to inflammation. Previous studies have shown that by cleaving ecDNA deoxyribonuclease (DNase) prevents the antibacterial effects of extracellular traps, but also has beneficial effects in sepsis. The aim of our study was to analyze the effects of DNase on WBCs in vitro and on ecDNA in a mouse model of sepsis. Our results confirmed that DNase decreases ecDNA by 70% and prevents the antibacterial effects of WBCs in vitro. Sepsis was induced in mice by intraperitoneal injection of E. coli. DNase was subsequently administered intravenously. In comparison to untreated septic mice DNase treatment improved the survival of septic mice by 60%, reduced their weight loss as well as inflammatory markers. Increased plasma DNase activity led to ecDNA concentrations in plasma comparable with the control group. In conclusion, the study showed that intravenous DNase improves survival of septic mice by cleavage of ecDNA, especially of nuclear origin. Further mechanistic studies are needed to prove the potential of DNase in the treatment or prevention of septic complications.