The present study includes energy, exergy and sustainability analyses of a diesel engine fueled with diesel and microalgae biodiesel (MB). The analyses were performed by using the results of experiments that were performed at different engine speeds (1500, 1800 and 2100 rpm). The results revealed that increasing engine speed enhanced the magnitudes of energy and exergy efficiencies of the engine fueled with diesel and MB. The energy efficiencies of the diesel engine fueled with diesel and MB at the engine speed of 2100 rpm were found to be 33.78% and 31.78%, while the corresponding exergy efficiencies were calculated to be 31.69% and 29.6 %, respectively. The highest sustainability index proportional to the exergy efficiency of the diesel engine was found at the 2100 rpm and these values for diesel and MB were found as 1.46 and 1.42, respectively. Moreover, the destructed exergy and the entropy generation showed an increase with increasing the engine speed due to the increase in thermodynamic irreversibilities. It can be also concluded that the findings of the analyses for the engine fueled with MB were found to be quite close to those of the engine fueled with diesel. MB can be used as a substitute of petroleum diesel.