BackgroundVector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namelyBabesia,Hepatozoon,Anaplasma phagocytophilumandBartonellain invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs.MethodsSpleen samples of 84 carnivores of eight species (Canis aureus,Canis lupus,Lynx lynx,P. lotor,Martes foina,Lutra lutra,Mustela ermineaandN. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA andcytBin hemoprotozoa,groELinA. phagocytophilum, and using multilocus genotyping inBartonellaspp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method.ResultsOut of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected inP. lotor, namelyBartonella canis,Hepatozoon canis,Hepatozoon martis,A. phagocytophilumandBartonellasp. related toBartonella washoensis. AllC. lupustested positive forH. canisand one forB. canis. Three VBPs (Hepatozoon silvestris,A. phagocytophilumandBartonella taylorii) were detected inL. lynxfor the first time.Babesia vulpesand yet undescribed species ofBabesia, not previously detected in Europe, were found inN. procyonoides.ConclusionsWild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones.Babesia vulpesand one new, probably imported species ofBabesia, were found in raccoon dogs.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05834-w.