Diphtheria incidence has dropped markedly thanks to global vaccination programs, yet some cases still occur even in highly vaccinated countries. We aimed to assess the seroprevalence of anti-diphtheria antibodies in the Italian population by age, sex, and geographical area.

Samples were collected from subjects (6–90 years) consulting for routine blood tests. Antibody levels were measured using an immuno-enzymatic assay (seroprotection threshold: 0.01  IU/ml).

Overall, 3,282 serum samples were collected in 2019–2020 from healthy individuals aged 6–90 years residing in 13 Italian regions. Overall, 81.4% of participants showed protective antibody levels. Protection decreased progressively with age: 90.7% among children aged 6–12, 85.4% in those aged 13–24, 78.6% in the 25–39 age group, and 73.2% among 40–64-year-olds. A slight increase was observed in individuals aged ≥ 65 (75.2%). High protection (≥0.1  IU/ml) was most prevalent in the youngest group (78.4%) and declined in older groups, reaching its lowest level in those aged 40–64 (44.7%). Conversely, low protection ( < 0.1  IU/ml) was highest in participants aged ≥ 65 (30.1%). Males showed significant higher protection than females in the 25–39, 40–64, and ≥ 65 age groups. Geographically, protection levels were higher in Northern (93.4%) and Central Italy (85.6%) than in Southern regions and the Islands (73.8%), for almost all age groups (except 6–12 years).

Protection against diphtheria in older age groups remains suboptimal. Booster vaccination strategies should be strengthened, as insufficient immunity in adults and the elderly poses a potential risk for severe disease outcomes.