Successful treatment of bacteremic patients depends largely on timely detection of blood-borne pathogens. Failure to detect an infection and/or contamination of blood samples can substantially delay the proper treatment. To increase the detection rate of blood-borne pathogens, well-established guidelines on blood collection and processing have been practiced in human medicine. Investigations involving human blood cultures have shown that the multiple blood sample approach significantly improves the detection rate of bacterial pathogens in the blood. Unfortunately, veterinary-specific blood culture guidelines have not been defined. Therefore, we compared detection rates of blood-borne pathogens between single and multiple blood culture approaches in a retrospective study of the clinical data from canine blood culture cases. We analyzed the data that had been collected over ~6 y and 8 mo from 177 dogs admitted to a veterinary medical teaching hospital. The triple blood culture approach increased the detection rate of blood-borne pathogens by 19.5% compared to single sampling. The optimal timing between multiple sample collections remains to be determined.