Information on the effects of a heterologous booster in adult patients first vaccinated with the BBIBP-CorV vaccine is limited. This prospective cohort study evaluated the humoral response of 152 healthcare workers (HCWs) from a private laboratory in Lima (Peru) before and after receiving the BNT162b2 vaccine, with a seven-month interval since the BBIBP-CorV doses. We employed the Elecsys® anti-SARS-CoV-2 S and the cPass™ SARS-CoV-2 Neutralization Antibody (NAbs) assays to evaluate anti-S-RBD IgG and NAbs, respectively. Of the 152 HCWs, 79 (51.98%) were previously infected (PI) with SARS-CoV-2 and 73 (48.02%) were not previously infected (NPI). The proportion of HCWs with positive NAbs, seven months after the BBIBP-CorV immunization, was 49.31% in NPI and 92.40% in PI. After the booster, this ratio increased to 100% in both groups. The anti-S-RBD IgG and NAbs in the HCWs’ NPI increased by 32.7 and 3.95 times more, respectively. In HCWs’ PI, this increment was 5 and 1.42 times more, respectively. There was no statistical association between the history of previous SARS-CoV-2 infection and the titer of anti-S-RBD IgG and NAbs after the booster. The humoral immunity presented a robust increase after receiving the BNT162b2 booster and was more pronounced in NPI.