Canine pathogens transmitted by blood-sucking arthropods are of significant importance for veterinary and, in some cases, human health. However, they are still underestimated and rarely investigated in many parts of the Mediterranean region, mostly due to financial reasons. Therefore, in the present paper, we investigated the occurrence of blood-associated pathogens affecting dogs in Crete, Greece. For this purpose, blood samples from 103 shelter dogs were screened for the pathogens by PCR and serological tests. Overall, samples from 43 dogs scored positive for at least one pathogen (41.8%). In particular, antibodies to Leishmania infantum were detected in 26 dogs (25.2%), and 15 and 11 animals were positive for Hepatozoon canis (14.6%) and Mycoplasma haemocanis (10.7%) by PCR, respectively. Co-infections were recorded in nine animals. Clinical signs indicative of infection (alterations of skin or coat or reduced body condition) were detected in 10 animals, four of which were infected with one pathogen, three with two pathogens. Based on the results obtained, dogs from Crete appear to be frequently exposed to several blood-borne pathogens, including agents of zoonotic concern. Given that some of the pathogens were reported for the first time in this area, results presented in our study should improve the awareness of the local veterinarians and of dog rescue organisations in order to reduce disease burden on stray and owned dogs and to control the spread of canine vector-borne diseases from Greece to non-endemic areas by travelling or exported infected dogs.