Health care workers (HCW) are a high-risk population to acquire SARS-CoV-2 infection from patients or other fellow HCW. This study aims at estimating the seroprevalence against SARS-CoV-2 in a random sample of HCW from a large hospital in Spain. Of the 578 participants recruited from 28 March to 9 April 2020, 54 (9.3%, 95% CI: 7.1–12.0) were seropositive for IgM and/or IgG and/or IgA against SARS-CoV-2. The cumulative prevalence of SARS-CoV-2 infection (presence of antibodies or past or current positive rRT-PCR) was 11.2% (65/578, 95% CI: 8.8–14.1). Among those with evidence of past or current infection, 40.0% (26/65) had not been previously diagnosed with COVID-19. Here we report a relatively low seroprevalence of antibodies among HCW at the peak of the COVID-19 epidemic in Spain. A large proportion of HCW with past or present infection had not been previously diagnosed with COVID-19, which calls for active periodic rRT-PCR testing in hospital settings.