Abstract Background As forest is the preferred environment for ticks, forestry workers are exposed to tick bites and tick-borne diseases. We assessed the seroprevalence of anti-Borrelia burgdorferi (Bb) antibodies and investigated, using an integrated landscape approach, the individual and environmental factors associated with the seroprevalence of Bb in Belgian forestry workers, a high-risk group in Belgium. Methods A group of 310 Belgian forest workers was examined to assess the seroprevalence of anti-Borrelia IgG antibodies. Using principal component analysis and binary logistic regression, the joint effects of individual characteristics and environmental characteristics were examined. Results Sixty-seven of the 310 workers were seropositive for Lyme disease (LD), leading to a seroprevalence of 21.6%. The seroprevalence was higher among forest workers visiting forests more frequently (P = 0.003) or who reported over 100 tick bites (P-value < 0.001). The intensity of tick bites and the use of protection measures against tick bites have a positive impact on LD seroprevalence while the quantity of shadow from trees at ground level had a negative one. Conclusions This study showed that forest workers are a population at risk for LD and, by extension, at risk for various tick-borne diseases. In addition to the role of the environment, our results also showed the importance of considering exposure when predicting the risk of infection by Bb.