A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty-six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA-based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR-positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram-negative cell wall, and a cytoplasmic membrane.