Co-infection with Borrelia species and Anaplasma phagocytophilum or Babesia spp. was assessed in a retrospective study of tick-exposed individuals from southeastern Poland. The co-infection rate of these pathogens was found to be rather low (Borrelia spp./Anaplasma phagocytophilum--4.2%, 1/24; Borrelia spp./Babesia spp.--4.2%, 1/24). However, due to the increased prevalence of Borrelia spp. in Ixodes ricinus ticks in Poland and the recent emergence of new tick-borne infections, it is necessary to carefully evaluate the true risk of human infection with several pathogens using more sensitive and reliable diagnostic tools. This is the first report of human infection with Babesia spp. in Poland that has been confirmed by molecular techniques with homology of 98.9% to B. divergens or Babesia EU1.