Introduction: Atherosclerosis is the major source of morbidity and mortality in the developed countries like Libya. Oxidative stress represents a risk factor and plays a key role at several steps of atherosclerosis. Formation of atherosclerotic plaques is the main reason for coronary artery disease. Aim: This study is aimed to assess the impact of the imbalance between body oxidants and antioxidants (oxidative stress) and its involvement in developing and severity of atherosclerosis in atherosclerotic with and without diabetes Libyan patients. Methods: The study sample comprised of atherosclerosis with and without diabetes patients (n = 73), as well as healthy controls (n = 27). Blood samples were collected to determine the levels of malonaldehyde (MDA) as oxidative biomarker, serum selenium level, total antioxidant status (TAS) level and some antioxidant enzymes including glutathione peroxidase (GPx), Catalase (Cat) and superoxide dismutase (SOD). Additionally, oxidant/antioxidant status was compared between atherosclerotic patients with and without diabetes in comparison to controls. Results: The results indicated a significant increase in MDA level among atherosclerotic patients compared to healthy subjects (P < 0.05). While the level of TAS, Cat, SOD and GPx was significantly decreased among patients compared to the controls (P < 0.05). For the atherosclerotic diabetic patients, the study was found a rise in the level of MDA as well as a marked decrease in TAS and the activity of tested antioxidant enzymes (P < 0.05). A good correlation was obtained between elevated MDA and decreased TAS, Cat, SOD and GPx. Conclusion: Determination of antioxidative defense markers contributes to understanding the effect of oxidative stress on the development and the prevention of cardiovascular disease.