The impact of the emerging tick-borne pathogen Borrelia miyamotoi is not fully understood. We utilised a protein array to investigate B. miyamotoi seroreactivity in various human populations in the Netherlands and Sweden. The IgM/IgG seroprevalence in Dutch healthy (2·5%, 95%CI 1·5–4·1) and population controls (2·0%, 95%CI 0·9–4·4) was lower (p = 0·01 and p = 0·01) compared to the tick-bite cohort (6·1%, 95%CI 3·9–9·5). In accordance, the Swedish healthy controls (1·0%, 95%CI 0·1–6·9) revealed a lower (p = 0·005 and p < 0·001) IgM/IgG seroprevalence compared to the tick-bite (8·9%, 95%CI 5·7–13·7) and fever after tick-bite cohort (16·5%, 95%CI 10·6–24·8). Altogether, 15 of 2175 individuals had serologic evidence of early B. miyamotoi infection. The risk of infection with B. miyamotoi was 0·7% (95%CI 0·3–1·4) in tick-bitten individuals, and of disease 7·3% (95%CI 2·6–12·8) in those with a febrile illness after tick-bite. Our findings provide insights into the risk of infection and disease with this pathogen in Europe.