To estimate the seroprevalence of Borrelia burgdorferi (Bb), Anaplasma phagocytophilum (Ap), and Francisella tularensis (Ft) in Belgium, we tested sera from three population-based samples in which exposure to pathogen is assumed to vary: 148 samples from workers professionally exposed, 209 samples from rural blood donors, and 193 samples from urban blood donors. Sera were tested using ELISA or the immunofluorescence assay test. The seroprevalence of Bb was 5.4% in workers professionally exposed, 2.9% in rural blood donors, and 2.6% in urban blood donors, which is similar to other studies. The fraction of negative results decreases significantly from urban blood donors and rural blood donors to workers. Regarding the seroprevalence of Ap, the cutoff titer of 1:64 recommended by the manufacturer may be set too low and produces artificially high seroprevalence rates. Using a cutoff titer of 1:128, the seroprevalence of Ap was estimated at 8.1% for workers professionally exposed, 6.2% for rural blood donors, and 5.7% for urban blood donors. Tularemia sera confirmed the presence of the pathogen in Belgium at 2.0% for workers and 0.5% for rural and urban blood donors. Our study is one of the few providing an estimation of the seroprevalences of Bb, Ap, and Ft in three different populations in Belgium, filling the gap in seroprevalence data among those groups. Our findings provide evidence that the entire Belgian population is exposed to Bb, Ap, and Ft infections, but a higher exposure is noticed for professionals at risk. Education on the risk factors for tick bites and preventive measures for both professionals exposed and the general population is needed.