Aim: This study aimed to compare dietary habits, obesity markers, and autonomic function among urban and rural populations to evaluate the impact of lifestyle differences on metabolic and cardiovascular health. Material and Methods: A comparative cross-sectional study was conducted among 200 participants, with 100 each from urban and rural areas. Participants were recruited from community health centers, educational institutions, and workplaces. Dietary intake was assessed using a validated Food Frequency Questionnaire (FFQ) and a 24-hour dietary recall. Anthropometric measurements, including body mass index (BMI), waist-hip ratio (WHR), and body fat percentage, were recorded. Autonomic function was evaluated using heart rate variability (HRV), blood pressure variability (BPV), and deep breathing tests. Statistical analysis was performed using SPSS version 25.0, applying chi-square tests, independent t-tests, Pearson and Spearman correlation, and multiple regression models. Results: Urban participants had significantly higher BMI (25.8 ± 3.2 kg/m² vs. 24.1 ± 3.0 kg/m², p = 0.015), WHR (0.85 ± 0.04 vs. 0.82 ± 0.05, p = 0.028), and body fat percentage (28.5 ± 5.1% vs. 26.3 ± 4.8%, p = 0.035) than rural participants. Physical activity levels were lower in urban participants (3200 ± 850 vs. 4500 ± 920, p < 0.001). Dietary intake analysis revealed higher fat consumption and lower fiber intake among urban participants, while rural participants had greater carbohydrate and protein intake. Autonomic function tests showed increased sympathetic dominance in urban participants, with a higher HRV LF/HF ratio (2.1 ± 0.5 vs. 1.8 ± 0.4, p = 0.018) and lower HRV RMSSD (32.5 ± 8.6 ms vs. 37.8 ± 7.9 ms, p = 0.009). Multiple regression analysis identified BMI, WHR, and carbohydrate intake as predictors of autonomic dysfunction, while physical activity had a protective effect. Conclusion: Urban participants exhibited higher obesity markers, lower physical activity, and greater autonomic dysfunction than rural participants. Dietary habits played a crucial role in modulating autonomic function, with high carbohydrate and fat intake linked to sympathetic dominance, while fiber and protein intake showed protective effects. These findings underscore the need for lifestyle interventions promoting balanced nutrition and increased physical activity to mitigate autonomic dysfunction and associated metabolic risks.