Recent reports suggest a potential for transmission of a newly discovered rickettsial endosymbiont, Midichloria mitochondrii, to animals and humans from feeding ticks (Ixodes ricinus). Using molecular methods; I. ricinus, sheep and red deer in Anaplasma phagocytophilum-endemic areas of Norway, were examined to see if they were infected by M. mitochondrii or related organisms like Wolbachia pipientis and Rickettsia spp. A total of 532 ticks collected from pastures, 76 blood samples from grazing lambs and 12 organ samples from hunted deer, were analyzed during the study. All larval pools, 60.4% pooled nymphs and 35.1% of adult ticks were positive for M. mitochondrii. There was a significant difference between geographical areas in the prevalence of M. mitochondrii infection among nymphs. A total of 2.2% pooled nymphs and 5.3% adult ticks were positive for A. phagocytophilum. Eleven percent of pooled nymphs were positive for Borrelia spp, 2.2% of pooled nymphs and 3.5% of adult ticks were positive for Rickettsia spp. and none of the ticks were positive for W. pipientis. The prevalence of A. phagocytophilum infection was 54% and 75% in grazing lambs and deer, respectively. No animals were positive for Borrelia spp., M. mitochondrii, Rickettsia spp. or W. pipientis. The reported findings suggest that M. mitochondrii is widespread in tick populations at different geographical sites, and may appear in co-infection with A. phagocytophilum, Borrelia spp. and Rickettsia spp. in ticks.