Cnicus benedictus fruits were used as raw material to extract oil, and the resulting oil was converted into biodiesel. Two extraction methods were tested: batch extraction, and ultrasound assisted extraction. Response surface methodology was considered for the optimization of the process efficiency. The selected key independent variables were temperature, extraction time, and solid/liquid ratio for batch extraction and ultrasound intensity, temperature, and extraction time for the ultrasound assisted extraction, respectively. The optimal working conditions are different for the two extraction techniques, with respect to temperature, solid/liquid ratio, and extraction time, respectively, leading to higher extraction efficiency in the case of the ultrasound-assisted extraction. Cnicus benedictus oil obtained under the optimal extraction conditions was further esterified with methanol under acid catalysis to yield biodiesel. The biodiesel was characterized through 1H-NMR and the main fuel properties were determined.