Sunflower oil is known for its therapeutic properties and culinary use. It is an important alimentary source of tocopherol and unsaturated fatty acids, and is used especially for wound healing. Studies on its antimicrobial potential, however, are lacking. The ozonation of oils of vegetable sources has been explored to enhance their therapeutic properties; however, studies that provide evidence of such benefits are still lacking. In the field of veterinary medicine, such data are even more scarce. In this study, the antimicrobial activity of ozonated sunflower oil was compared to that of non-ozonated oil, in an in vitro system, against strains of Staphylococcus aureus and Escherichia coli, isolated from intrauterine lavages of mares with endometritis. Tests were conducted using the minimum inhibitory concentration method. The ozonated oil was effective against S. aureus, whereas it was not against E. coli isolates. Our data open doors for discussion on the use of sunflower oil, with or without ozone treatment, for therapeutic purposes in veterinary medicine.