The use of diesel-farnesane and sugarcane biodiesel is showing significant potential for reducing harmful emissions from Brazilian road transport. Both biofuels are obtained from sugarcane through synthetic biology that requires fermentation. The present work studies the effect of the sugarcane biofuels, compared to regular diesel fuel, on the performance and emissions of a modern medium-duty diesel engine following the European Steady Cycle (ESC) test procedure. Diesel-farnesane showed the lowest particulate matter (PM) specific emissions. Specific nitrogen oxides (NOx) emissions from diesel fuel were above the standard limit, while sugarcane biodiesel produced the lowest emissions. All tested fuels produced specific carbon monoxide (CO) emissions below the standard limit, but the biofuels showed higher values than the reference fuel. Sugarcane biodiesel showed an inhibition effect on the oxidation process at the Diesel Oxidation Catalyst (DOC), leading to the highest specific CO emissions after the DOC. Specific total hydrocarbons (THC) emissions were well below the standard limits for all tested fuels. Concerning the regulated emissions for diesel engines (g/h), the use of sugarcane biodiesel led to PM and NOx reductions of 32.4% and 37.3%, while diesel-farnesane led to 41.7% and 6.08% reductions, respectively. These notable reductions in harmful emissions support the application of sugarcane biofuels to road transport.