The feasibility of testing Garden cress Lepidium sativum L. seed as a beneficial reference for biodiesel (BD) creation as an outcome of its satisfactory oil content (28.50± 1.50 % w/w) was explored in detail. The alkali-catalyzed alcoholysis reaction of the garden cress seed oil (GCSO) with ethanol and mixed methanol/ethanol alcohols was investigated with determining the optimum reaction conditions. Under the typical conditions of the reaction, the maximum yields of ethylic BD and mixed methylic/ethylic BD were 90.45 ± 2.0 % and 97.50± 1.50%, respectively. The FTIR, 1H NMR, and thin-layer chromatography studies assured the transformation of GCSO to the expected BD fuel. The elementary characteristics of the BD samples besides BD-petro diesel mixtures were determined and found compatible with specifications set by ASTM criteria. The alcoholysis reaction of the GCSO obeyed the first-order kinetics with activation energy values of 30.71 KJ/mol for the ethanolysis of the GCSO, and 32.93 KJ/mol for the transesterification with mixed methanol/ethanol alcohols. This study disclosed that the GCSO possessed the potential to be utilized as a successful alternative to petroleum-based diesel.