Abstract This epizootiological study was carried out to investigate the occurrence of canine babesiosis in southwestern Slovakia. The study focused on the proportion of the species of ticks serving as babesia vectors of babesiosis in the ticks collected from selected locations close to Komárno, in southwestern Slovakia. Additionally, observations were made on the health and overall clinical signs in dogs suspected of having babesiosis. In 2014 we collected ticks from vegetation by the drag cloth (flagging) method and also directly from dogs. A totally of 622 ticks were collected: 491 Dermacentor reticulatus and 131 Ix-odes ricinus. Representative samples of ticks (n = 103) were examined by the molecular method and the presence of Babesia spp. DNA was identified in 12.5 % of the Ixodes ricinus ticks collected by flagging and in 9.5 % of the Ixodes ricinus ticks collected from the dogs. Babesia canis (KU681325) with 90 bp sequence, 100 % identical with Babesia canis isolates from dogs for example from: Turkey (KF499115), Rumania (HQ662634), Croatia (FJ209025), Poland (EU622792) and Russia (AY962186), was confirmed after sequencing in one Ixodes ricinus female obtained from a dog. This was the first confirmation of the occurrence of B. canis in Ixodes ricinus ticks in Slovakia. In 2.2 % of the Dermacentor reticulatus ticks obtained from vegetation by flagging, we were able to diagnose the DNA of Babesia canis. In 4.8 % of the Dermacentor reticulatus ticks collected from dogs, the presence of Babesia spp. was confirmed. Thirty three dogs with suspicion of babesiosis were observed in an ambulance by their health and clinical signs. The loss of appetite was observed in 22 patients (66.7 %), apathy in 19 cases (57.6 %), and fever in 19 cases. Closer specification indicating babesiosis was finding engorged ticks on the dog bodies which occurred in 21 cases (63.6 %), haematuria in 8 cases (24.2 %), anaemia in 4 cases (12.1 %), and tremor in 6 cases (18.2 %). Other non-specific signs, such as diarrhoea, vomitus, slowed-down movement, ataxia and lacrimation were also observed in less than 18 % of the examined dogs. On the basis of the specific clinical signs, blood was withdrawn from 33 dogs for preparation of blood smears and in 19 of them (57.6 %) babesia in erythrocytes were confirmed microscopically.