Healthcare workers (HCWs) are a vulnerable and critical population in the ongoing response to the SARS-CoV-2 pandemic. We aimed to estimate the seroprevalence in HCWs considering all of their previous contacts with the SARS-CoV-2 virus and/or the immunity acquired through their immunization against COVID-19 before the advent of the Omicron variants BA.4/BA.5. Serum samples were collected from 28 March to 10 June 2022. We covered 25% out of all the people who worked in some of the government healthcare centers (primary, secondary, and tertiary level) across the entire Autonomous Province of Vojvodina (Northern Serbia). Two serological tests (Anti-SARS-CoV-2 QuantiVac ELISA and LIAISON® SARS-CoV-2 TrimericS) were used to detect anti-spike IgG antibodies. The overall prevalence of the SARS-CoV-2 antibody among the 6936 HCWs was 92.96% [95% CI 92.33–93.55]. Regarding the type of serological test, there was a statistically significant (p = 0.0079) difference of the seropositivity obtained by the LIAISON® SARS-CoV-2 TrimericS (93.87%, 95% CI 92.97–94.69) and Anti-SARS-CoV-2 QuantiVac ELISA (92.23%, 95% CI 91.34–93.06) tests. Seropositivity to SARS-CoV-2 significantly (p < 0.0001) increased with the number of SARS-CoV-2 infections combined with the number of doses of the SARS-CoV-2 vaccines received. A vast majority of the HCWs in Vojvodina had detectable levels of antibodies to the spike protein of SARS-CoV-2, but despite this high seropositivity, it is unknown whether this herd immunity among HCWs is protective against the new variants of concern. Further research should evaluate the rates of reinfections and the associated severity of COVID-19 caused by the Omicron sublineages and/or new variants of SARS-CoV-2 among HCWs.