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Background: SARS-CoV-2 is a novel human pathogen causing Coronavirus Disease 2019 that has caused widespread global mortality and morbidity. Since health workers in Israel were among the first to be vaccinated, we had a unique opportunity to investigate the post-vaccination level of IgG anti-S levels antibodies (Abs) and their dynamics by demographic and professional factors. Methods: Prospective Serological Survey during December 2020–August 2021 at Barzilai Medical Center among 458 health care workers (HCW) followed for 6 months after the second BNT162b2 vaccine dose. Results: Antibody levels before the second dose, and 30, 90 and 180 days after were 57.1 ± 29.2, 223 ± 70.2, 172.8 ± 73.3 and 166.4 ± 100.7 AU/mL, respectively. From GEE analysis, females had higher Abs levels (β = 26.37 AU/mL, p = 0.002). Age was negatively associated with Abs, with a 1.17 AU/mL decrease for each additional year (p < 0.001). Direct contact with patients was associated with lower Abs by 25.02 AU/mL (p = 0.009) compared to working with no such contact. The average decline rate overall for the study period was 3.0 ± 2.9 AU/mL per week without differences by demographic parameters and was faster during the first 3 months after vaccination than in the subsequent 3 months. Conclusions: All demographic groups experienced a decline in Abs over time, faster during the first 3 months. Findings of overall Abs lower in males, workers with direct contact with patients, and older workers, should be considered for policy-making about choosing priority populations for additional vaccine doses in hospital settings.