Introduction: Grassroots health workers are crucial in Vietnam's healthcare system. As the frontline of healthcare, closest to the community, they are considered the backbone for disease prevention, control efforts, and initial healthcare for the population. The Ministry of Health in Vietnam has endeavored to improve infrastructure, equipment, and workforce quality, along with providing mental health care for grassroots healthcare workers to effectively carry out health education, disease prevention, and well-organized healthcare services. This paper aims to investigate factors affecting depression, anxiety, and stress among grassroots health workers in An Giang Province located in the Vietnamese Mekong Delta region. Methods: A population-based cross-sectional study was conducted using data from the 2023 Grassroots health workers in An Giang Province. A total of 466 grassroots health workers were selected. The Research method to collect quantitative data from the questionnaire tools was applied and data analysis was conducted using the SPSS 22.0. Results: The research findings showed that 18.3%, 28.8%, and 16.8% of participants exhibited symptoms of depression, anxiety, and stress, respectively, predominantly at mild, moderate, severe, and extremely severe levels. Risk factors impacting mental health included income and benefits, COVID-19 infection, and prolonged symptoms affecting physical health, as well as workplace infrastructure. Conclusions: Improving salary policies, physical health care, and upgrading workplace infrastructure will reduce the risk of mental health issues among grassroots health workers, both during the pandemic and in the future.