ObjectivesHealthcare workers (HCWs) have been one of the most severely affected groups during the COVID-19 pandemic, though few studies have sought to determine the rate of undiagnosed cases among this population. In this study, we aim to determine the rate of undetected infection in HCWs, a potential source of nosocomial infection.MethodsSerological screening for IgG and IgM antibodies against SARS-CoV-2 was carried out among HCWs from four different hospitals in Madrid, Spain, from 6 April to 25 April 2020; HCWs with a previous diagnosis of infection based on real-time reverse transcriptase-PCR assay performed after presenting compatible symptoms were excluded. Prevalence of IgG and IgM antibodies was calculated among HCWs to obtain the rate of COVID-19 presence of antibodies in each hospital.ResultsOf the 7121 HCWs studied, 6344 (89.09%) had not been previously diagnosed with COVID-19. A total of 5995 HCWs finally participated in the study, resulting in a participation rate of 94.49%. A positive IgM or IgG test against COVID-19 was revealed in 16.21% of the HCWs studied (n=972).ConclusionThis study reveals the importance of early detection of SARS-CoV-2 infection among HCWs to prevent nosocomial infection and exposure of patients, visitors and workers and the spread of COVID-19 in the overall community.