Ixodes ricinusandDermacentor reticulatusticks are important reservoirs and vectors of pathogens. The aim of the present study was to investigate the dynamic of the prevalence and genetic diversity of microorganisms detected in these tick species collected from two ecologically diverse biotopes undergoing disparate long-term climate condition. High-throughput real time PCR confirmed high prevalence of microorganisms detected in sympatrically occurring ticks species.D. reticulatusspecimens were the most often infected withFrancisella-like endosymbiont (FLE) (up to 100.0%) andRickettsiaspp. (up to 91.7%), while in case ofI. ricinusthe prevalence of Borreliaceae spirochetes reached up to 25.0%. Moreover, pathogens belonging to genera ofBartonella,Anaplasma,EhrlichiaandBabesiawere detected in both tick species regardless the biotope. On the other hand,Neoehrlichia mikurensiswas conformed only inI. ricinusin the forest biotope, while genetic material ofTheileriaspp. was found only inD. reticulatuscollected from the meadow. Our study confirmed significant impact of biotope type on prevalence of representatives of Borreliaceae and Rickettsiaceae families. The most common co-infection detected inD. reticulatuswasRickettsiaspp. + FLE, while Borreliaceae +R. helveticawas the most common inI. ricinus. Additionally, we found significant genetic diversity ofR. raoultii gltAgene across studied years, however such relationship was not observed in ticks from studied biotopes. Our results suggest that ecological type of biotope undergoing disparate long-term climate conditions have an impact on prevalence of tick-borne pathogens in adultD. reticulatusandI. ricinus.