BackgroundNewly recognized endemic foci for human babesiosis include Europe, where Ixodes ricinus, a vector for several species of Babesia, is the most commonly identified tick. Vector-based surveillance provides an early warning system for the emergence of human babesiosis, which is likely to be under-reported at emerging sites. In the present study, we set out to screen I. ricinus collected from Danish domestic dogs for Babesia, in order to identify whether humans in Denmark are exposed to the parasite.FindingsA total of 661 ticks (Ixodes spp.) were collected from 345 Danish domestic dogs during April-September 2011 and pooled, one sample per dog. DNA was extracted from each sample and examined by PCR and sequencing for Rickettsia spp., Borrelia burgdorferi sensu lato, Bartonella spp., Francisella tularensis, Candidatus Neoehrlichia mikurensis, and Babesia spp. In total, 34% of the samples were positive for tick-borne microorganisms potentially pathogenic to humans: Rickettsia spp. were detected in 16% of the pools, with 79% being R. helvetica. Borrelia burgdorferi sensu lato was found in 15%, with the main species identified as Borrelia afzelii (39%). Likewise, 8% of the samples were positive for Babesia spp. (Babesia microti, 82%; Babesia venatorum (‘EU1’), 18%). Lastly, 1% of the samples tested positive for Candidatus Neoehrlichia mikurensis, and 0.6% for Bartonella spp. No ticks were found to be infected with Francisella tularensis.ConclusionsOur data are in support of endemic occurrence of potentially zoonotic Babesia in Denmark and confirms I. ricinus as a vector of multiple pathogens of public health concern.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0843-0) contains supplementary material, which is available to authorized users.