3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 μM and 150 μM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.