Mannosylerythritol lipid-B (MEL-B) is a glycolipid whose biological properties have been widely investigated, especially in the skincare, food, and therapeutic fields. Despite this, few studies have addressed the toxicity of this glycolipid in vivo. Therefore, this work aimed to evaluate the in vivo oxidative stress induced by MEL-B in Swiss mice. MEL-B (50 and 150 mg/kg) was administered intraperitoneally at two exposure times, 24 and 72 h. Biochemical damage was quantified in the gastrocnemius, lungs, kidneys, heart, liver, and spleen. This study assessed the levels of reactive oxygen species, oxidative damage markers, antioxidant defenses, protein concentration, triglycerides, creatine kinase (CK-MB), and lactate dehydrogenase (LDH). DCF (2',7'-dichlorofluorescein), sulfhydryl, and SOD (superoxide dismutase) levels were used to assess oxidative damage and antioxidant defenses in cells. The results indicate that MEL-B did not trigger acute toxicity in the tested animals in a systemic context. Oxidative stress was observed in the liver samples, likely due to the metabolization of MEL-B. The levels of triglycerides and of CK-MB and LDH enzymes did not present any significant alteration (