We provide here an updated analysis of an ongoing serosurveillance study, presenting data on the effect of a third dose of Pfizer/BioNTech BNT162b2 vaccine on serum anti-SARS-CoV-2 IgG antibodies. We tested baseline SARS-CoV-2 seronegative healthcare workers undergoing primary vaccination with the mRNA-based COVID-19 Comirnaty vaccine, followed by administration of homologous vaccine booster (third dose). Venous blood was collected before either dose of primary vaccination, at 1, 3 and 6months afterwards, as well as before and 1month after receiving the vaccine booster. The serum concentration of anti-SARS-CoV-2 IgG was assayed with DiaSorin Trimeric spike IgG immunoassay. The final study population included 53 SARS-CoV-2 seronegative healthcare workers (median age 46years; 60% females). A first peak of anti-SARS-CoV-2 spike trimeric IgG values was reached 1month after completing primary vaccination, after which the levels gradually declined until before receiving the vaccine booster. A second peak of anti-SARS-CoV-2 spike trimeric IgG concentration was observed 1month after receiving the vaccine booster dose (8,700kBAU/L), which was 39-fold higher than before receiving the vaccine booster (221kBAU/L; p<0.001), but was also nearly threefold higher compared to values seen at the first peak (2,990kBAU/L; p<0.001). The rate of subjects with protective anti-SARS-CoV-2 spike trimeric IgG values (i.e.,>264kBAU/L) increased from 47.2% to 100% after 1month from vaccine booster. These results support current policies fostering COVID-19 vaccine boosters to reinforce humoral immunity against SARS-CoV-2.