The purpose of this study was evaluation of the prevalence of co-infection with Borrelia species, A. phagocytophilum and Babesia spp. in patients with tick-borne encephalitis (TBE). At total of 110 patients with TBE were included in the study. Serological tests for tick-borne encephalitis virus (TBEV), PCR for Borrelia species, Anaplasma phagocytophilum and Babesia spp., blood smears for A. phagocytophilum and Babesia spp. and BLAST analysis for Babesia spp. were performed. Results showed a significant majority of patients co-infected with Borrelia species (30/110; 27 %), much less with A. phagocytophilum (12/110; 10.9 %) and with Babesia spp. (1/110; 0.9 %). The BLAST analysis of the 18S rDNA sequence obtained with the Babesia spp. specific primers indicated that the patient was infected with Babesia microti. Triple co-infections (TBEV-Borrelia species- A. phagocytophilum) were observed in three (3/110; 2.7 %) patients. Conclusions were such that differential diagnosis in patients after the tick bite, presenting with acute symptoms, should include not only TBE and Lyme disease, but also other diseases transmitted by ticks. In patients with low parasitemia in suspicion of Babesia spp. infection PCR seems to be a more sensitive method than blood smear. Co-infection with various tick-borne pathogens must be always considered, especially in endemic regions.