This study shows which hard tick species (Ixodidae) were found on domestic horses in the Netherlands in 2008–2009, and what potential pathogens these ticks carried. In the period 2008–2009, 130 ticks were collected, classified and screened for the presence of DNA from specific tick-borne pathogens using PCR-RLB. The numbers of ticks of the various species found were: 68 Ixodes ricinus, 58 Ixodes spp. (57 nymphs and 1 larva), 2 Dermacentor reticulatus and 2 Hyalomma marginatum. DNA from Borrelia valaisiana was detected in 49% of these ticks, B. afzelii in 22%, B. burgdorferi sensu stricto and B. garinii in 3% and 2%, respectively. Rickettsia helvetica was detected in 9% of examined ticks, Anaplasma phagocytophilum in 1.5%, Babesia venatorum in 4%, and B. caballi and Theileria equi in 1.5 and 3%, respectively. There were considerable regional differences suggesting focal distribution of these potential pathogens.