The determination of the cytotoxic effects of compounds which are used in cancer treatment for chemotherapeutic and chemopreventive purposes on target cells as well as on healthy cells have oriented the researchers to the studies aiming to reduce the treatment-induced toxicity. Therefore, the studies about the effectiveness of plant sources in cancer treatment have gained importance. In this study, cytotoxic effects of aqueous extract and essential oil of Origanum minutiflorum O. Schwarz et. P.H. Davis on canine mammary tumor cell lines and Swiss 3T3 albino mouse fibroblast cell line were determined by MTT assay. Also, for the determination of antioxidant activity and total phenolic content of extract and essential oil 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and Folin-Ciocalteu reagent were used, respectively. Cytotoxic effects of essential oil obtained from the Origanum minutiflorum O. Schwarz et. P.H. Davis were observed on canine mammary tumor cell lines in a concentration dependent manner (86.67-7.51% and 74.45-2.42%). Despite this, cytotoxic effect of the essential oil was observed only at the highest concentration (200 μg/ml) on Swiss 3T3 albino mouse fibroblast cells, a healthy cell line. It has been determined that DPPH free radical scavenging activity of the essential oil and extract were close to each other, while total phenolic content of the essential oil was higher than the extract. The results have indicated that the essential oil extracted from Origanum minutiflorum O. Schwarz et. P. H. Davis has shown selective toxic effect on canine mammary tumor cells.