Understanding the long-term determinants of antibody levels against SARS-CoV-2 is crucial for evaluating population immunity and guiding public health strategies. While short- and mid-term immune responses after COVID-19 vaccination have been extensively studied, data on long-term humoral immunity remain limited. This observational study was conducted at G. Gennimatas General Hospital of Thessaloniki, Greece. A total of 104 healthcare workers with varying vaccination and infection histories were included to identify the key factors influencing antibody levels. Participants had received three or more doses of the Pfizer-BioNTech mRNA COVID-19 vaccine (BNT162b2, Comirnaty), except for 11 individuals who had received only two doses. Serum samples were collected three years after the third vaccine dose between October 2024 and November 2024. Antibody levels increased after the second and third vaccine doses and subsequently declined over time. In a multivariable regression analysis adjusting for age, sex, number of vaccine doses, and number of infections, we showed that the most important determinant of antibody levels was the individualspecific time (in days) elapsed since the last immunological event, either vaccination or infection. Other factors, including demographic characteristics and cumulative exposure to the virus or vaccines, had no significant independent effect when accounting for time. These findings suggest that waning immunity is the primary driver of antibody levels, emphasizing the need for periodic booster vaccinations to maintain protection in healthcare workers.  Kulcsszavak: BNT162b2 mRNA COVID-19 vaccine (Comirnaty), Greece, SARS-CoV-2, antibody titers, healthcare workers