From May until October 2007, a total of 658 Ixodes ricinus ticks were collected off birds (189), rodents (273), and vegetation (196) in a certain area of Middle Germany and investigated for infection with Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Overall, 13.1% (86/658) of the ticks were infected with at least one pathogen; co-infections occurred in 0.6% (4/658). Babesia spp. specific DNA was detected in 9.7% (64/658) of the ticks, 1.4% (9/658) were infected with A. phagocytophilum, and 2.6% (17/658) harbored rickettsiae. At least two different Rickettsia species were identified: Rickettsia monacensis and Rickettsia helvetica. Our study provides first interesting insights into the circulation and co-circulation of several emerging pathogens not only in ticks parasitizing birds and small mammals as potential reservoirs but also in questing ticks in a single natural habitat.