The COVID-19 pandemic in Brazil affected mental health among healthcare workers. To objective of this study was to evaluate the mental health of healthcare workers in in the central-west region of the Brazil, estimating the prevalence of mental health disorders, and investigating associated factors, perceptions of safety, and self-perceptions about mental health in times of the COVID-19 pandemic. The questionnaire was divided into two parts that included general information and perceptions about the work process and identified symptoms using the Depression Anxiety Stress Scale-21 (DASS-21), and multiple linear regression analysis was conducted. A total of 1,522 healthcare workers participated in the survey. Overall prevalence of symptoms was calculated for depression (58.7%), anxiety (59.7%), and stress (61.7%). Physicians had 3.75 times greater risk of depression (1.59–8.85, 95% CI). Independent variables associated with depression symptoms were not feeling safe with the way services were organized (1.12:1.03–1.21, 95% CI) and self-perception of poor mental health (8.06: 4.03–16.10% CI). Working in management was protective, and married professionals had 12% lower risk of exhibiting symptoms of depression (0.79–0.99, 95% CI). Participants with self-perception of poor mental health had 4.63 greater risk for symptoms of anxiety (2.58–8.31, 95% CI). Protective factors were not having sought support for mental health (0.90: 0.82–0.99, 95% CI), having a graduate degree (0.71: 0.54–0.94, 95% CI), and not having been diagnosed with COVID-19 (0.90: 0.83–0.98, 95% CI). Perception of poor mental health was associated with 6.95-fold greater chance of developing stress symptoms. Protective factors from stress were having a degree in dentistry (0.81: 0.68–0.97, 95% CI), residing in Mato Grosso do Sul (0.91: 0.85–0.98, 95% CI), and not having sought mental health support services (0.88: 0.82–0.95, 95% CI). The prevalence of mental health disorders is high among healthcare workers, and is associated with professional category, organization of services provided, and self-perception of poor mental health, reinforcing the need for preventative measures.