Lyme borreliosis (LB) is not notifiable in many European countries, and accurate data on the incidence are often lacking. This study aimed to determine the seroprevalence of Borrelia burgdorferi sensu lato (s.l.)-specific antibodies in the general population of The Netherlands, and to determine risk factors associated with seropositivity. Sera and questionnaires were obtained from participants (n = 5592, aged 0–88 years) enrolled in a nationwide serosurveillance study. The sera were tested for B. burgdorferi s.l.-specific IgM and IgG antibodies using ELISA and immunoblot. Seroprevalence was estimated controlling for the survey design. Risk factors for seropositivity were analyzed using a generalized linear mixed-effect model. In 2016/2017, the seroprevalence in The Netherlands was 4.4% (95% CI 3.5–5.2). Estimates were higher in men (5.7% [95% CI 4.4–7.2]) than in women (3.1% [95% CI 2.0–4.0]), and increased with age from 2.6% (95% CI 1.4–4.4) in children to 7.7% (95% CI 5.9–7.9) in 60- to 88-year-olds. The seroprevalence for B. burgdorferi s.l. in the general population in The Netherlands was comparable to rates reported in European countries. The main risk factors for seropositivity were increasing age, being male and the tick bite frequency. The dynamics of LB infection are complex and involve variables from various disciplines. This could be further elucidated using infectious disease modelling.