Background: The COVID-19 pandemic has placed an unprecedented physical and mental burden on healthcare workers who are frequently at high risk of infection, particularly in low-income countries. This study aimed to assess the prevalence and associated factors of anxiety, depression, and stress, as well as changes in daily and occupational activities among healthcare professionals due to the COVID-19 pandemic in Colombia.Methods: An observational, cross-sectional study was conducted between February and June 2021. The survey incorporated validated mental health tools such as the Generalized Anxiety Disorder–7, the Patient Health Questionnaire-9, and the Perceived Stress Scale-10. Multivariable ordinal logistic regression analysis was performed to determine the factors associated with severe mental health outcomes.Results: Among 1345 healthcare workers the prevalence of anxiety, depression, and stress were 75.61%, 59.18%, and 53.09%, respectively. Anxiety (OR:1.44; 95%CI:1.16-1.8), depression (OR:1.74; 95%CI:1.27-2.37), and stress (OR:1.51; 95%CI:1.18-1.94) were more frequent in women, and individuals who expressed fear of a negative outcome (death, sequelae) (OR:2.25; 95%CI:1.60-3.25), (OR:1.49; 95%CI:1.03-2.16) and (OR:2.36; 95%CI:1.69-3.29) respectively. Age was negatively associated with anxiety (OR:0.98; 95%CI:0.98-0.99), stress (OR:0.98; 95%CI:0.97-0.99), and depression (OR:0.97; 95% CI:0.96-0.98). Reduction in consultations and surgeries (OR:1.01; 95%CI:1.0-1.01) was positively associated with anxiety. Due to the pandemic, most specialists expected to incorporate drastic long-term (>1 year) changes in their clinical setting and daily activities.Conclusions: The prevalence of anxiety, depression, and stress is higher among Colombian healthcare workers compared to previous reports. Further research regarding these psychological outcomes is needed to achieve early mental health intervention strategies.Trial Registration: Hospital Universitario Fundación Santa Fe, Ethical Committee Registration ID: CCEI-12992-2021.