Aim: The aim of this study was to evaluate the comparative activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in smokers versus non-smokers and to assess the associated oxidative stress markers. Materials and Methods: A total of 120 participants, including 60 smokers and 60 non-smokers aged 25-50 years, were selected for this study. Venous blood samples were collected to measure the activity of antioxidant enzymes (SOD, CAT, GPx) using spectrophotometric assays. Oxidative stress markers, including malondialdehyde (MDA) and total antioxidant capacity (TAC), were also analyzed. Statistical analysis was performed using independent t-tests. Results: Significant reductions in antioxidant enzyme activity were observed in smokers compared to non-smokers (SOD: 1.82 ± 0.29 U/mg vs. 2.34 ± 0.31 U/mg, CAT: 34.5 ± 4.6 U/mg vs. 41.2 ± 5.1 U/mg, GPx: 18.7 ± 2.8 U/mg vs. 22.9 ± 3.0 U/mg, all p < 0.001). In contrast, oxidative stress markers were elevated in smokers, with MDA levels significantly higher (5.8 ± 0.9 nmol/mL vs. 3.1 ± 0.7 nmol/mL) and TAC lower (910 ± 102 µmol/L vs. 1150 ± 130 µmol/L, p < 0.001). A negative correlation between smoking duration, intensity, and antioxidant enzyme activity was also observed. Conclusion: Chronic smoking significantly impairs antioxidant defenses and increases oxidative stress. The results underscore the importance of smoking cessation and lifestyle modifications, including improved diet and physical activity, to mitigate oxidative damage and reduce the risk of smoking-related diseases.