Purpose This study aims to determine the seroprevalence of coronavirus disease (COVID-19) among health care workers and describe the associated sociodemographic and labor features. Patients and Methods An observational study with an analytical component was conducted at a clinic in Cali, Colombia. The sample size was 708 health workers and they were selected by stratified random sampling. A Bayesian analysis was developed to determine the raw and adjusted prevalence. A Poisson regression model was used to estimate the prevalence ratios. Results Overall seroprevalence of COVID-19 among healthcare workers was 29%. Miscellaneous services workers, healthcare, and administrative workers, was 38%, 33%, and 32%, respectively. Factors related to seropositivity were having a contact with a COVID-19 patient for >120 minutes and being diagnosed with COVID-19 by laboratory tests. Conclusion The present study shows a adjusted seroprevalence of 29% in health workers, indicating a high level of disease transmission and an increased risk of infection in this population group.