The effects of extra virgin olive oil (EVOO) and carbon tetrachloride (CCl4) induced oxidative stress in rats were determined by the generation of isoprostanoids. These are known to be robust biomarkers to evaluate nonenzymatic and free radical related oxidation. Other oxidative stress biomarkers such as hydroxyeicosatetraenoic acid products (HETEs) and cholesterol oxidation products (COPs) were also determined. The rodents received a control diet, high-fat diet (20% w/w) composed of extra virgin olive oil (EVOO), corn oil (CO), or lard, and high-fat diets with CCl4 insult throughout the experimental period. The EVOO diet was found to suppress the formation of isoprostanoids and COPs compared to that of the control. EVOO also had a high total phenolic content and antioxidant activity compared to those of CO and lard and may be contributed to by the hydroxytyrosol component conjugated to fatty acids (HT-FA). This is the first study to identify HT-FA in EVOO, and it was 4-fold higher than that of olive oil, whereas none was found in corn oil. Furthermore, the EVOO diet showed reduced liver lipid vesicles in CCl4 treated rats compared to that of the control. However, liver toxicity measurements of AST (aspartate transaminase) and ALT (alanine transaminase) activities showed augmentation with CCl4 treatment but were not alleviated by the diets given. Our findings suggest that EVOO is a daily functional food capable of enhancing the antioxidant system for liver protection; the effect is potentially attributed to the phenolic and lipophenolic (phenol conjugated by fatty acids) content.