Lyme borreliosis (LB) is not notifiable in many European countries, and the patchwork of surveillance strategies in Europe perpetuates knowledge gaps. In the Netherlands, LB incidence has been estimated from recurring general practitioner surveys since the 1990s. To complement the incidence data, this study aimed to estimate the prevalence of antibodies against Borrelia burgdorferi sensu lato in the general population of the Netherlands in 1995/1996, identify risk factors for seropositivity, and compare these findings to data from 2016/2017 to identify temporal trends. Sera from participants (n = 8041, aged 0–80 years) in a cross-sectional nationwide surveillance study were assessed for the presence of antibodies against B. burgdorferi s.l., using a screening ELISA and immunoblot confirmation. Risk factors associated with seropositivity were evaluated using multivariable analysis. A significant difference in weighted seroprevalence was observed between 1995/1996 (2.8%) and 2016/2017 (4.3%). In both cohorts, the seroprevalence was significantly higher among men than among women, and increased with age and tick bite frequency. The upward trend in age-specific seropositivity in individuals over 50 was steeper in 2016/2017 than in 1995/1996, possibly due to improved fitness among contemporary elderly, allowing increased outdoor activities. This study highlights significant trends in the seroprevalence of B. burgdorferi s.l. antibodies in the general population of the Netherlands over 20 years. The doubling of seroprevalence underscores the increasing burden of LB, and the importance of continued surveillance. Targeted interventions, particularly for elderly populations, may help raise awareness to the risks of tick bites and reduce the growing disease burden and societal costs associated with LB.
