ABSTRACT Neutralizing antibodies (NTAb) play a significant role in preventing and protecting against SARS-CoV-2 virus infection. Identifying NTAb is undoubtedly imperative in understanding the immunity toward COVID-19 better. However, it is interesting to note that the production of NTAb varies among individuals, especially among healthcare workers (HCWs), as they are exposed to the virus daily. Hence, we would like to investigate factors affecting the production of S-RBD IgG and NTAb among different categories of HCWs, particularly after receiving the third dose of the BNT162b2 mRNA COVID-19 Vaccine. A total of 361 HCWs from our hospital were prospectively enrolled and had their S-RBD IgG and NTAb titers measured. They were studied in relation to the degree of exposure to COVID-19, breakthrough infections, gender, age, race, household income, housing type, household number, and education levels. HCWs with the highest risk of exposure to COVID-19, breakthrough infections, and male gender displayed the highest median titers of both S-RBD IgG and NTAb, and the differences were statistically significant (p < .05). Age, race, household income, housing type, household number, and education levels were revealed to be insignificant. We concluded that the degree of exposure to COVID-19, breakthrough infections, and male gender are significant factors in NTAb production among HCWs.