Smokeless and smoking tobacco use results in increased oxidative stress and lipid peroxidation, which play a major role in the causation of cancer in tobacco habituates. Malondialdehyde (MDA) is a product of lipid peroxidation, and glutathione peroxidase (GPx) and superoxide dismutase (SOD), the main enzymes in the antioxidant defense system, are assessed among tobacco users. This study gave insight into the relationship between tobacco use, oxidative stress, and antioxidant enzyme activity. This study aimed to estimate the levels of lipid peroxidation product MDA and antioxidant enzymes SOD and GPx among tobacco users and compare them with controls. A case-control study comprising 30 smokeless tobacco users, 30 smokers, and 30 controls was enrolled for the study. Serum MDA was assayed by the thiobarbituric acid method; serum SOD and GPx were assayed using Ransel antioxidant kits. The results were statistically analyzed using descriptive and inferential statistical analysis. Serum MDA levels, which indicate oxidative stress, were increased among all tobacco users and significantly increased among smokeless tobacco users as compared to smokers. Serum SOD and GPx levels were decreased among both forms of tobacco users compared with controls. With an increase in duration and frequency of tobacco use, there was a significant increase in serum MDA levels among both smokers and chewers and a decrease in serum SOD and GPx levels. In the present day, the tobacco epidemic has attained enormous proportions with the tobacco habit starting as early as 13-14 years and leading to serious conditions with high morbidity and mortality. These biochemical parameters such as MDA, SOD, and GPx, which act as marker of oxidant and antioxidant system, can constitute important tools for evidence-based medicine for educating patients and motivating interventions in tobacco cessation therapy.