Sepsis is characterized by a dysregulated immune response to infection characterized by an early hyperinflammatory and oxidative response followed by a subsequent immunosuppression phase. Although there have been some advances in the treatment of sepsis, mortality rates remain high, urging for the search of new therapies. β-Lapachone (β-Lap) is a natural compound obtained from Tabebuia avellanedae Lorentz ex Griseb. with several pharmacological properties including bactericidal, anti-inflammatory, and antioxidant activity. Thus, the aim of this study was to evaluate the effects of β-Lap in a mouse sepsis model. To this, we tested two therapeutic protocols in mice submitted to cecal ligation and puncture- (CLP-) induced sepsis. First, we found that in pretreated animals, β-Lap reduced the systemic inflammatory response and improved bacterial clearance and mouse survival. Moreover, β-Lap also decreased lipid peroxidation and increased the total antioxidant capacity in the serum and peritoneal cavity of septic animals. In the model of severe sepsis, the posttreatment with β-Lap was able to increase the survival of animals and maintain the antioxidant defense function. In conclusion, the β-Lap was able to increase the survival of septic animals by a mechanism involving immunomodulatory and antioxidant protective effects.