The study was conducted to detect Brucella sp. and Leptospira sp. in blood samples of dogs in Isfahan and Shahrekord province in Iran. A total of 94 blood samples were collected from dogs of different breed, age, sex, and dogs' type (stray or nonstray). The samples were examined using conventional polymerase chain reaction (PCR). Fourteen (14.89%) dogs were positive for Brucella sp. and 18 (19.15%). dogs for Leptospira sp. There were no significant differences between the prevalence of the pathogens, provinces, sex, and age groups (P > 0.05). However, there was a statistically significant difference in prevalence of Brucella sp. and Leptospira sp. between stray and non-stray dogs (P < 0.0001; χ2 = 30.3767). The study also demonstrated that PCR was successfully used for the first time in Iran for the detection of Brucella sp. and Leptospira sp. in blood samples of dogs. Therefore, we recommend the PCR as a supplementary method with other commonly recognised methods (e.g. serological methods) for the diagnosis of subclinical infections with the microorganisms. Strict measures for the control of stray dogs are also highly recommended.