Introduction <p>Ambulance drivers play a vital role in providing emergency services. These tasks render their job physically, mentally, and emotionally taxing due to prolonged and irregular working hours.</p> Objectives <p>The primary objectives of the study were to estimate the prevalence of depression, anxiety, and stress among ambulance drivers in Tamil Nadu and to identify the factors affecting these conditions among these drivers.</p> Methods <p>A cross-sectional study design was employed using the Depression, Anxiety and Stress Scale 21 (DASS-21) among 385 ambulance drivers to estimate the levels of depression, anxiety, and stress in two major cities of Tamil Nadu, Chennai and Coimbatore.</p> Results <p>Among the 385 participants, 16% of ambulance drivers exhibited severe to extreme symptoms of depression, whereas 48% displayed severe to extreme symptoms of anxiety. Furthermore, 11% indicated encountering severe or extreme levels of occupational stress. Factors including exposure to violence during duty, participation in training with involvement in lifesaving, length of sleep, and nature of services (road traffic accidents versus non-road traffic accidents) substantially affected the intensity of symptoms associated with anxiety, stress, and depression.</p> Conclusion <p>Due to the high-risk nature of their profession and their role as emergency service providers, ambulance drivers require an adequate support system to improve their mental well-being.</p>