Abstract Introduction The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) booster vaccination campaign and the emergence of SARS-CoV-2 Omicron variants impact the prevalence and levels of SARS-CoV-2 antibodies in the Netherlands. In this study we determined antibody levels across age groups, the impact of Omicron variant infections, and the effect of booster vaccinations on antibody levels. Methods In September and December 2021 and in February 2022, over 2000 Dutch blood donors were tested for presence of SARS-CoV-2 antibodies. Donations were selected based on age, sex, and region of residence, to provide an optimal coverage and representation of the Dutch population. Results Levels of vaccination-induced spike antibodies decreased over time in all age groups. Donors vaccinated with Janssen or AstraZeneca had significantly lower antibody levels than donors vaccinated with Pfizer or Moderna vaccine. Boostering with an mRNA vaccine elevated antibody levels in all age-groups irrespective of the initial vaccine. In donors aged < 56 years, the proportion of infected donors almost doubled between December 2021 and February 2022. Conclusion The booster vaccination campaign increased antibody levels in all age-groups. After a booster vaccination, donors initially vaccinated with AstraZeneca or Janssen vaccine showed antibody levels similar to donors initially vaccinated with an mRNA vaccine. The emergence of the SARS-CoV-2 Omicron variant in the Netherlands caused a substantial increase in donors with infection-induced antibodies, especially among younger donors.