ObjectivesThis study aimed to assess the prevalence of blood‐borne infectious agents in healthy, client‐owned dogs from a blood donor population in Portugal and Spain, and to address the importance of a screening protocol on every donation.Materials and MethodsClient‐owned healthy dogs were tested before each donation on a veterinary blood bank. Blood samples from new potential donors, and from regular donors, were tested by real‐time polymerase chain reaction for Leishmania species, Ehrlichia species, Brucella species, Babesia species and Anaplasma species Serological tests were also performed for Leishmania species, Ehrlichia species and Dirofilaria immitis. All donors were tested for every infectious agent in each donation.ResultsThe study found that out of a total of 8036 donors and 35,120 samples tested, 3.9% of blood donors tested positive for at least one of the agents, with the most prevalent being Anaplasma species (2.1%). Serological tests also revealed positive results in 14.0% of donors, with the highest percentage for Leishmania species (7.7%). Moreover, the study found that 28.2% of positive results were from dogs with negative results in donations performed 3 to 12 months before, and 18.0% of positive results were recent infections.Clinical SignificanceThese findings indicate a high prevalence of infectious agents in seemingly healthy, selected dogs eligible to become blood donors in the Iberian Peninsula, highlighting the importance of regular testing on every donation. This study emphasises the importance of a regular screening protocol for every donation instead of annual testing, as is commonly performed in veterinary medicine.