The development of biofuels from oleaginous biomass has attracted a great deal of interest not only because of the environment pollution but also because of the rapid depletion of the fossil fuel reserves. Among the biomass conversion processes, the solvolysis reaction has shown to be an interesting solution which allows the direct conversion of raw materials into biofuel. In the present work, a solvolysis process is applied on Pistacia lentiscus (PL) seeds using ethanol as solvent for bio-oil production. Several temperatures varying from 270 to 330 °C, reaction time in the range 15–45 min and ethanol:PL seed ratio values from 0.5–1.5 ml/g were considered. The response surface methodology (RSM) combined with Box-Behnken design (BBD) is used for modeling and optimizing both the process yield and bio-oil viscosity. The results show that the optimum conditions are obtained for a reaction temperature of 304.91 °C, a reaction time of 35.24 min, and an ethanol:PL seed ratio of 1.16. Under these conditions, the predicted PL bio-oil yield is around 63.5% with a bio-oil viscosity of 5.88 mm2/s. The PL bio-oil physicochemical properties showed a good agreement with those of diesel fuel and in conformity with the international requirements except for the viscosity, whose value was found to be slightly higher than the standard. The engine tests show that the fuel specific consumption increases around 9% with a mixture containing bio-oil up to 30%. On the other hand, slight increases in CO (13%), HC (25%) and particulate (30%) emissions were recorded.