The combination of lower concentrations of antitumor drugs (carboplatin – CARB, doxorubicin – DOX, and methotrexate – MET) with propolis was investigated against canine osteosarcoma (spOS-2) and mesenchymal stem cells (MSC) in vitro. The mechanism of action in the combinations was analyzed. spOS-2 cells were incubated up to 72 h with propolis (50 μg/ml) alone or in combination with CARB (10–400 μmol/l), DOX (0.5–2 μmol/l) or MET (50–200 μmol/l). Cell viability was assessed by MTT assay, apoptosis/necrosis by flow cytometry, and MSC was incubated with the optimum combination. Propolis alone exerted no cytotoxic action against spOS-2 cells, whereas CARB (400, 200 and 100 μmol/l) exhibited the highest cytotoxic effects comparing to DOX and MET. The combination of propolis with the lowest concentrations of CARB led to better results comparing to CARB alone, which was not observed using DOX and MET. Apoptosis was involved in the action of propolis + CARB in spOS-2 cells. MSC were not affected by CARB/propolis, indicating that the cytotoxic action of the combination was specific to tumor cells but not to normal ones. Propolis improved the action of CARB against spOS-2 cells using lower concentrations of this drug, without affecting MSC. These findings are relevant and indicate a possible application of propolis in OSA treatment.