Biodiesel is an alternative diesel fuel produced from animal and vegetable oils. As an alternative and ecologically acceptable substitute for conventional fuel, biodiesel is produced from a wide variety of edible vegetable oils that are usually used for human consumption and whose prices are expected to rise in the future. In this context, reliable and lowcost raw materials are gaining increasing interest for biodiesel production, such as by-products of meat processing industries or waste animal fats. Biodiesel production from waste animal fat, and raw food does not compete with the industry and has a great potential for waste caused by the global decline. In our study, a potential alternative fuel was produced for diesel engines by using the non-food-grade fat portion of horse meat consumed in Middle Asia countries. Solid crude horse oil was liquefied, and its fatty acid components were analyzed and transformed into horse oil biodiesel by the transesterification method. It was determined whether the fuel properties of crude horse oil, horse oil biodiesel, and euro diesel fuel comply with the standard values, and their usability in diesel engines was investigated. As a result of the tests, it has been concluded that horse oil biodiesel does not meet the standards in terms of cold flow properties and can only be used at a rate of low volumetric ratios in diesel engines. This article will contribute to the use of horse oil biodiesel production stages and fuel properties in diesel engines and future studies.