The preparation of a butanol/butyl butyrate mixture was performed in one-step under continuous flow conditions with a CuO/ZrO₂ catalyst. The catalytic system allows one to directly obtain up to 40–42% of butyl butyrate starting from butanol via a dehydrogenative coupling reaction without using solvent or additives. The obtained mixture was tested in a direct injection spark ignition engine as a blend of 70%_vol gasoline and 30%_vol butanol/butyl butyrate mixture. One of the main goals was to evaluate overall performance and whether knock tendency increased compared to the reference condition that featured gasoline only fueling. Exhaust gas pollutants were evaluated as well, so as to give a more complete picture of environmental impact effects. Overall engine performance and emissions were found to be comparable to those obtained for the reference case, with negligible increase in knocking characteristics.