Altogether, 430 nymphs and 570 adult Ixodes ricinus ticks were collected in 2006 (n = 506) and 2007 (n = 494) from a forest area in Middle Germany (Thuringia). Single ticks were investigated by polymerase chain reaction and restriction fragment length polymorphism or sequencing for Borrelia spp. (ospA gene) and Babesia spp. (18S rRNA gene). Overall, 27.0% (270/1000) were infected with Borrelia species. Out of these, Borrelia garinii was detected most frequently (133/270)-especially, OspA serotype 6 (51/270), followed by Bo. burgdorferi (70/270), Bo. afzelii (42/270), Bo. valaisiana subgroup I (28/270), not typable Borrelia spp. (5/270), Bo. spielmanii (3/270), Bo. valaisiana subgroup II (2/270), and Bo. lusitaniae (1/270). In 1.4% of investigated ticks mixed infections with several Borrelia spp. occurred. Babesia spp.-specific DNA was detected in 5.0% of ticks. Babesia microti was slightly more prevalent (28/50) than Babesia divergens (20/50). Moreover, 5.9% (16/270) of Borrelia spp.-infected ticks were coinfected with Babesia spp. Knowledge on the degree of heterogeneity of Borrelia species and OspA types is prerequisite not only for local risk assessment, but also for diagnostic test and vaccine development.