Objectives. To assess the baseline prevalence of mental health conditions and associated exposures in a cohort of health care workers (HCWs) in Guatemala. Methods. We analyzed baseline information from the 2020 Web-based COVID-19 Health Care Workers Study (HEROES)-Guatemala. Outcomes included mental distress and depressive symptoms. Exposures included COVID-19 experiences, sociodemographic characteristics, and job characteristics. We used crude and adjusted Poisson regression models in our analyses. Results. Of the 1801 HCWs who accepted to participate, 1522 (84.5%) completed the questionnaire; 1014 (66.8%) were women. Among the participants, 59.1% (95% confidence interval [CI] = 56.6, 61.5) screened positive for mental distress and 23% (95% CI = 20.9, 25.2) for moderate to severe depressive symptoms. COVID-19 experiences, sociodemographic characteristics, and job characteristics were associated with the study outcomes. Participants who were worried about COVID-19 infection were at higher risk of mental distress (relative risk [RR] = 1.47; 95% CI = 1.30, 1.66) and depressive symptoms (RR = 1.51; 95% CI = 1.17, 1.96). Similarly, the youngest participants were at elevated risk of mental distress (RR = 1.80; 95% CI = 1.24, 2.63) and depressive symptoms (OR = 4.58; 95% CI = 1.51, 13.87). Conclusions. Mental health conditions are highly prevalent among Guatemalan HCWs. (Am J Public Health. 2022;112(S6):S602-S614. https://doi.org/10.2105/AJPH.2021.306648).