Bovine trypanosomoses, caused by Trypanosoma vivax, is a disease present in African and South American countries. This haemoflagellate protozoan parasite, as well as Anaplasma marginale and Babesia spp., are microorganisms that have a blood tropism, mainly causing fever and anaemia, which reduces the productive capacity of dairy or meat farms. This study aimed to detect T. vivax and other blood parasites in bovine herds in the Galapagos Islands. A total of 170 blood samples from bovines in 19 farms on Santa Cruz Island (the most populated) were collected and analyzed using different PCR techniques: Da-PCR and CatL-PCR to detect Trypanosoma vivax, CatL-PCR to detect Trypanosoma theileri, ESAG-PCR to detect Trypanosoma evansi, 18S rRNA-PCR to detect Babesia spp., rap-1-PCR to detect Babesia bovis, hyp-PCR to detect Babesia bigemina, and msp5-PCR to detect A. marginale. The prevalence of T. vivax, B. bovis, B. bigemina, and A. marginale was estimated as 14.7%, 11.2%, 14.7%, and 67.1%, respectively. In this study, the presence of four haemotropic agents was evidenced in 26.3% (5/19) of the farms. Coinfected cattle (A. marginale, B. bovis and B. bigemina) had significantly higher body temperatures compared to others (two-sample Wilcoxon rank-sum test; p-value = 0.047). The molecular techniques used in this study demonstrated the presence of T. vivax and B. bovis in cattle from Santa Cruz Island in the Galapagos for the first time. The study also investigates the relationship between T. vivax, A. marginale and Babesia spp., making a significant contribution to the field of veterinary medicine.