They are described two blend fuels with a high hydrotreated vegetable oil (HVO) share and two sustainable biofuels of the fatty acid methyl ester and HVO types of the ZETOR 9540 tractor in the framework of a long-term operational test. Compared to standard fossil diesel, the results of measuring performance parameters and characteristics for the assessment of the use of these fuels tractors and other agricultural machinery are mentioned. It was specified the quality, manufacturer, emission factors and greenhouse gas savings of clean certified biofuels and their high percentages of fossil diesel. For samples from all tested fuels, comprehensive fuel analysis was performed in accordance with the technical standards.