Because a large proportion of the Austrian population has been infected with SARS-CoV-2 during high incidence periods in winter 2021/2022, up-to-date estimates of seroprevalence of anti-SARS-CoV-2 antibodies are required to inform upcoming public health policies. We quantified anti-Spike IgG antibody levels in 22,607 individuals that donated blood between October 2021 and April 2022 across Tyrol, Austria (participation rate: 96.0%). Median age of participants was 45.3 years (IQR: 30.9–55.1); 41.9% were female. From October 2021 to April 2022, seropositivity increased from 84.9% (95% CI: 83.8–86.0%) to 95.8% (94.9–96.4%), and the geometric mean anti-Spike IgG levels among seropositive participants increased from 283 (95% CI: 271–296) to 1437 (1360–1518) BAU/mL. The percentages of participants in categories with undetectable levels and detectable levels at <500, 500–<1000, 1000–<2000, 2000–<3000, and ≥3000 BAU/mL were 15%, 54%, 15%, 10%, 3%, and 3% in October 2021 vs. 4%, 18%, 17%, 18%, 11%, and 32% in April 2022. Of 2711 participants that had repeat measurements taken a median 4.2 months apart, 61.8% moved to a higher, 13.9% to a lower, and 24.4% remained in the same category. Among seropositive participants, antibody levels were 16.8-fold in vaccinated individuals compared to unvaccinated individuals (95% CI: 14.2–19.9; p-value < 0.001). In conclusion, anti-SARS-CoV-2 seroprevalence in terms of seropositivity and average antibody levels has increased markedly during the winter 2021/2022 SARS-CoV-2 waves in Tyrol, Austria.