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Background: Healthcare workers are at increased risk of SARS-CoV-2 infection. The positivity rates in hospitals that do not receive patients with COVID-19, such as the National Cancer Institute (INCan) in Mexico, and the associated factors are unknown., Objective: To assess the incidence and factors associated with SARS-CoV-2 infection in health workers at INCan., Methods: A cohort study of 531 workers who were followed for 6 months. RT-PCR analysis of saliva and nasopharyngeal swab samples were used in the baseline and to confirm cases during follow-up The incidence rate ratio was calculated according to the measured characteristics and the associated factors were calculated using logistic regression models., Results: Out of 531 workers, 9.6% tested positive for SARS-CoV-2, Being male (RR: 2.07, 95% CI: 1.1-3.8, P = .02), performing administrative tasks (RR: 1.99, 95% CI: 1.0-3.9, P = .04), and having relatives also working at INCan (RR: 3.7, 95% CI: 1.4-9.5, P < .01) were associated with higher positivity rates., Discussion: Incidence of positive cases in health workers were similar to that reported in non-COVID hospitals from other countries., Conclusions: Even though active surveillance helped to detect a significant number of asymptomatic infections, it is still necessary to reinforce preventive measures in non-medical staff to prevent nosocomial transmission., (Copyright © 2021 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)