The development of renewable and sustainable candidates for petroleum-based fuels is needed to address the present issue of the increasing fuel crisis regarding transportation, environmental pollution, and consumption of the petroleum reserves. In addition, there is a need to explore new non-edible oils for biodiesel production due to problems such as fuel versus food as well as procurement and presence. Therefore the experimental research was carried out to synthesize biodiesel from Styrax officinalis L. oil. It has been firstly investigated and reported as a novel feedstock for the production of alternative fuel. The raw material was subjected to esterification at desired reaction parameters estimated by 9-runs (L9) orthogonal approach of Taguchi technique. Noteworthy, the oil content was found to be at 48.29 +/- 3.81%. The maximum biodiesel yield of 89.23% was obtained under the following optimized conditions: catalyst concentration of 0.6 wt%, methanol to oil molar ratio of 6:1, reaction duration of 60 min and reaction temperature of 60 degrees C. The detection of the substantial parameters was achieved using the Taguchi method and the significant parameters were obtained as follows: catalyst concentration, methanol to oil molar ratio, reaction duration, and reaction temperature with contribution factors of 78.07%, 20.32%, 0.42%, and 1.19%, respectively. Statistical analysis employing ANOVA exhibited that emerged outcomes are in good agreement with the predicted values. The fuel properties of the methyl esters from Styrax officinalis L. oil were within the ranges of the EN 14214 specifications. Therefore, the novel seeds can be a suitable feedstock for biodiesel production in the nearest future.