This work investigates a diesel engine operating with different blends of diesel fuel, biodiesel and anhydrous ethanol. Anhydrous ethanol (99.8% purity) was added to diesel oil with 7% (B7) and 20% of biodiesel (B20), with the concentrations of 5% (E5), 10% (E10) and 15% (E15) and 20% (E20). The experiments were conducted on a naturally aspirated, four-stroke, four-cylinder, direct injection 44 kW diesel engine, operating at a constant speed of 1800 RPM and at the fixed load of 27.5 kW to attain the lowest specific fuel consumption (SFC). The results were compared with the standard B7 operation, and showed that ethanol addition (B7E5) reduced up to 7% carbon dioxide (CO2) emissions, associated with the decrease of the cylinder gas temperature, due to the ethanol high latent heat of evaporation, and to the ethanol lower carbon-to-hydrogen ratio and oxygen content. Total hydrocarbons (THC) emissions were reduced up to 14% with ethanol addition (B7E15), indicating higher fuel burn efficiency when ethanol is added to the fuel, as the oxygen available in ethanol molecule improves the burning during combustion. On the other hand, increasing biodiesel content in the fuel from 7% to 20% increased CO2 and THC emissions, both mitigated with the use of ethanol. Carbon monoxide (CO) and oxides of nitrogen (NOX) emissions showed different behavior, depending on ethanol and biodiesel concentration. Both biodiesel and ethanol increased SFC, due to the reduction of fuel lower heating value (LHV), although ethanol addition slightly increased fuel conversion efficiency.