This study has determined oil, fatty acid (FA) and phytosterols content during the ripening of the Tunisian Onopordum acanthium L. seeds. In total, nine FAs and six phytosterols were identified. The main FAs were linoleic acid (0.18–8.06 mg/g of seed) followed by oleic acid (0.051–2.45 mg/g of seed), palmitic acid and stearic acid. Pentadecanoic acid was detected, for the first time, in unripe fruits and the two last stages of development were characterised by a relative abundance of erucic acid. Overall, β-sitosterol (34.5–77.79% of total sterols) was the major 4-desmethylsterols during maturation. The first episodes of growth were characterised by the best amounts of stigmasterol and campesterol, while stigmastanol and Δ7 sitosterol had quoted the semi-ripe and fully ripe fruits; however, cholesterol was absent. These findings are useful in understanding a potential new source of important natural compounds (Phytosterols and USFA) found in this fruit and when harvest should be undertaken to optimise desired FA and phytosterols content.