This study aimed to determine the cumulative incidence, prevalence, and seroconversion of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its associated factors among healthcare workers (HCWs) of a University Hospital in Bogotá, Colombia. An ambispective cohort was established from March 2020 to February 2021. From November 2020 to February 2021, SARS-CoV-2 antibodies were measured on two occasions 14-90 days apart to determine seroprevalence and seroconversion. We used multivariate log-binomial regression to evaluate factors associated with SARS-CoV-2 infection. Among 2,597 HCWs, the cumulative incidence of infection was 35.7%, and seroprevalence was 21.5%. A reduced risk of infection was observed among those aged 35-44 and ≥45 years (adjusted relative risks [aRRs], 0.84 and 0.83, respectively), physicians (aRR, 0.77), those wearing N95 respirators (aRR, 0.82) and working remotely (aRR, 0.74). Being overweight (aRR, 1.18) or obese (aRR, 1.24); being a nurse or nurse assistant (aRR, 1.20); working in the emergency room (aRR, 1.45), general wards (aRR, 1.45), intensive care unit (aRR, 1.34), or COVID-19 areas (aRR, 1.17); and close contact with COVID-19 cases (aRR, 1.47) increased the risk of infection. The incidence of SARS-CoV-2 infection found in this study reflects the dynamics of the first year of the pandemic in Bogotá. A high burden of infection calls for strengthening prevention and screening measures for HCWs, focusing especially on those at high risk.