There is uncertainty about the seroprevalence of anti-SARS-CoV-2 antibodies in the general population of Austria and about the waning of antibodies over time. We conducted a seroepidemiological study between June 2020 and September 2021, enrolling blood donors aged 18–70 years across Tyrol, Austria (participation rate: 84.0%). We analyzed serum samples for antibodies against the spike or the nucleocapsid proteins of SARS-CoV-2. We performed a total of 47,363 samples taken from 35,193 individuals (median age, 43.1 years (IQR: 29.3–53.7); 45.3% women; 10.0% with prior SARS-CoV-2 infection). Seroprevalence increased from 3.4% (95% CI: 2.8–4.2%) in June 2020 to 82.7% (95% CI: 81.4–83.8%) in September 2021, largely due to vaccination. Anti-spike IgG seroprevalence was 99.6% (95% CI: 99.4–99.7%) among fully vaccinated individuals, 90.4% (95% CI: 88.8–91.7%) among unvaccinated individuals with prior infection and 11.5% (95% CI: 10.8–12.3%) among unvaccinated individuals without known prior infection. Anti-spike IgG levels were reduced by 44.0% (95% CI: 34.9–51.7%) at 5–6 months compared with 0–3 months after infection. In fully vaccinated individuals, they decreased by 31.7% (95% CI: 29.4–33.9%) per month. In conclusion, seroprevalence in Tyrol increased to 82.7% in September 2021, with the bulk of seropositivity stemming from vaccination. Antibody levels substantially and gradually declined after vaccination or infection.