BACKGROUND: The longitudinal study was conducted over the initial 2 years of the COVID-19 pandemic, spanning from June 2020 to December 2022, in healthcare workers (HCWs) of the Thomayer University Hospital. A total of 3892 blood samples were collected and analyzed for total nucleocapsid (N) antibodies. The aim of the study was to evaluate the dynamics of N antibodies, their relationship to the PCR test, spike (S) antibodies, interferon-gamma, and prediction of reinfection with SARS-CoV-2. METHODS: Blood collections were performed in three rounds, along with questionnaires addressing clinical symptoms of past infection, PCR testing, and vaccination. Antibody measurements included total N antibodies (Roche Diagnostics) and postvaccination S antibodies (Euroimmun). Cellular immunity was tested by interferon-gamma release assay (Euroimmun). RESULTS: At the end of the study, 35.9% of HCWs were positive for N antibodies, and 39.5% of HCWs had either known PCR positivity or N antibodies or both. Ten percent of participants had no knowledge of a COVID-19 infection and 35% of positive individuals exhibited no symptoms. The values of positive antibodies decrease over a period of 6 months to 1 year, depending on the initial value, and their dynamics are highly variable. The study also demonstrated that the highest levels of spike antibodies and interferon-gamma occur during so-called hybrid immunity. CONCLUSION: Nucleocapsid antibodies proved valuable in monitoring SARS-CoV-2 infection dynamics, and they may detect cases of SARS-CoV-2 infection missed by PCR tests. The study identified distinct patterns in antibody dynamics and protection of hybrid immunity during reinfection.