This study investigated the protective properties of garlic essential oil (GEO) and its major organosulfur component (diallyl disulfide, DADS) against the development of nonalcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a normal or high-fat diet (HFD) with/without GEO (25, 50, and 100 mg/kg) or DADS (10 and 20 mg/kg) for 12 weeks. GEO and DADS dose-dependently exerted antiobesity and antihyperlipidemic effects by reducing HFD-induced body weight gain, adipose tissue weight, and serum biochemical parameters. Administration of 50 and 100 mg/kg GEO and 20 mg/kg DADS significantly decreased the release of pro-inflammatory cytokines in liver, accompanied by elevated antioxidant capacity via inhibition of cytochrome P450 2E1 expression during NAFLD development. The anti-NAFLD effects of GEO and DADS were mediated through down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as stimulation of peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase-1. These results demonstrate that GEO and DADS dose-dependently protected obese mice with long-term HFD-induced NAFLD from lipid accumulation, inflammation, and oxidative damage by ameliorating lipid metabolic disorders and oxidative stress. The dose of 20 mg/kg DADS was equally as effective in preventing NAFLD as 50 mg/kg GEO containing the same amount of DADS, which demonstrates that DADS may be the main bioactive component in GEO.