Purslane (Portulaca oleracea L.) is a widespread weed plant used since ancient times as remedy and as food. Its seed oil possesses significant bioactive potential due to the high content of important phytonutrients, mainly essential fatty acids and phytosterols. Although the total fatty acids composition of oil is well documented, till now there is no data published about individual lipid classes. The information on sterols is scarce as well. Therefore, the aim of this work was to characterize in details for the first time the individual lipid classes and their fatty acids composition, sterols, as well as the oil oxidative stability, depending on the extraction method by either hexane, chloroform-methanol mixtures or super-critical CO2. The results revealed no significant effect of extraction method on the individual lipid classes (monoacylglycerols, diacylglycerols, free fatty acids, free sterols, triacylglycerols, sterol esters, wax esters and hydrocarbons), including the fatty acids composition of saponifiables, and quite weak effect on the oil oxidative stability regarding super-critical CO2 vs. organic solvents. Detailed analyses of lipid composition confirmed the potential of purslane seed oil as a cheap and highly valuable source of phytonutrients as essential fatty acids and phytosterols, for application in food, pharmaceutical and cosmetic industries.