Microbial oil (SCO) is called lipids accumulated in the cells of oleaginous microorganisms, including yeast, in amount exceeding 20% of dry mass, which are a valuable source of fatty acids in the human diet. In order to facilitate the extraction of storage lipids from cells, methods of physical and chemical pretreatment of biomass are used to break the barrier of the cell wall and membrane of these microorganisms to the action of organic solvents, which are used during traditional extraction. The aim of the study was to evaluate the effectiveness of unconventional methods of extracting microbial oil from Yarrowia lipolytica yeast cells. Pulsed electric field (PEF), cell disintegration by ultrasonic waves and high-pressure homogenization (HPH) were used. The use of unconventional methods turned out to be ineffective in the extraction of intracellular lipids of the yeast compared to methods involving organic solvents such as chloroform, methanol and hexane. Nevertheless, the use of a pulsed electric field with a field strength of 200 J/g or high-pressure homogenization (700 and 1100 bar) proved to be effective as pre-treatment techniques of Y. lipolytica yeast cells (cell permeabilization) for the high yield extraction of intracellular lipids using the extraction method with organic solvents.