The reported cases of pertussis vary considerably globally. In the present nationwide study, we aimed to record the Bordetella pertussis prevalence in Greece by measuring serum IgG specific antibody levels to pertussis toxin (anti-PT IgG). General practitioners and laboratories participated in this study from 12 regions of Greece. A geographically stratified sampling plan based on regional units (NUTS level 2) was applied in order to produce a representative sample, taking into consideration age group (30–39, 40–49, 50–59, 60–69, 70–79 and 80+) and sex. In total, 1169 subjects participated in the study. The percentage of participants with anti-PT IgG antibodies higher than 50 IU/mL was 3.7%. The levels of anti-PT IgG antibodies of total sample ranged between 1.46 IU/mL to 126.60 IU/mL, with mean 17.74 IU/mL and standard deviation 14.03 U/mL (p-value 90%, suggesting that a large number of adults may be vulnerable to infection of pertussis despite well-established vaccination programs in Greece. Despite the fact that vaccination reduced the number of reported pertussis cases in the last decades in Greece, our seroprevalence study may indicate that the herd immunity level among Greek adults is suboptimal.