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Background To assess SARS-CoV-2 seroprevalence in healthcare workers (HCW) with sampling in June and October 2020 and April and November 2021. Methods Observational and prospective study in 2455 HCW with serum sampling. Antibodies against SARS-CoV-2 nucleocapsid and occupational, social and health risk factors were assessed at each time point. Results Seropositivity for SARS-CoV-2 in HCW increased from 11.8% in June 2020 to 28.4% in November 2021. Of those with a positive test in June 2020, 92.1% remained with a positive test, 6.7% had an indeterminate test and 1.1% had a negative test in November 2021. Non-diagnosed carriers represented 28.6% in June 2020 and 14.6% in November 2021. Nurses and nursing assistants showed the highest prevalence of seropositivity. Close contact (at home or in the hospital) with Covid-19 cases without protection and working in the frontline were the main risk factors. A total of 88.8% HCW were vaccinated, all with a positive serological response in April 2021, but levels of antibodies decreased about 65%, and two vaccinated persons presented a negative serological test against spike protein in November 2021. Levels of spike antibodies were higher in those vaccinated with Moderna compared with Pfizer and the percentage of antibody reduction was higher with Pfizer vaccine. Conclusions This study shows that seroprevalence of SARS-CoV-2 antibodies among HCW doubled that of the general population and that protection both at the workplace and in the socio-familial field was associated with a lower risk of infection, which stabilized after vaccination.