This study aimed to identify the phytochemical compounds of Matricaria pubescens by LC-MS/MS and evaluate the potential protective effect of its supplementation in high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in adult rats through modulation of oxidative stress and histopathological changes. Twenty-four male rats were randomly divided into four groups. The first group served as control and received the standard diet. The second group (HFD) received a high-fat diet only (30 % of sheep fat). The third group's (control+MP) animals received a standard diet supplemented with 5 % M. pubescens (w/w). The fourth group (HFD+MP) received a high-fat diet supplemented with 5 % M. pubescens for 16 weeks. LC-MS/MS analysis showed that M. pubescens contains many phytochemical compounds. It was observed that the ethanolic extract of M. pubescens has a higher phenolic content than the aqueous extract. The supplementation of M. pubescens (5 % w/w) to HFD rats decreased significantly (p<0.01) body weight, liver and epididymal adipose tissue relative weights, glycemia, triglycerides (TG), insulin resistance, liver markers, TNF-α, malondialdehyde (MDA), protein carbonyl (PCO), advanced oxidation protein products (AOPP) level, and increased reduced glutathione (GSH) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase activities as well as ameliorated histological alterations through the reduction hepatic lipid deposition and adipocytes hypertrophy compared to the HFD group. We conclude that M. pubescens powder may be effective for correcting hyperglycemia, hypertriglyceridemia, insulin resistance, and liver markers while decreasing inflammation and oxidative stress in the liver of high-fat diet-fed rats.