The SARS-CoV-2 pandemic led to the development of various vaccines. The BNT162b2 mRNA vaccine was the first approved due to its efficacy in eliciting a humoral immunity response after the second dose. However, a decrease in the antibody concentration was observed over time. Therefore, the administration of a third dose was scheduled, primarily for frail people and workers of essential public activities. The aim of this study was to assess the level of antibodies against the spike (S) RBD of SARS-CoV-2 in healthcare workers before and after the third dose of BNT162b2 vaccine, according to sex, age, and the time interval between vaccine doses and tests. All 37 (12 males, 25 females, 19 p < 0.05). This study indicated the benefit of the third dose of BNT162b2 vaccine and its effect on leveling up the humoral immune response.