<p>The aim of this study was to evaluate different cell disruption techniques, the efficiency of intracellular lipid extraction using alternative solvents and to characterize the microbial oil extract from yeast <i>Yarrowia lipolytica</i> NRRL Y-1095. Acid hydrolysis (HCl 5&#xa0;mol L⁻¹) at 80&#xa0;°C for 1&#xa0;h was the most effective technique for cell disruption, with a lipid yield of 17.45 ± 1.06% (w w⁻¹). Lipid extractions using the solvent systems ethyl acetate/ethanol/water and hexane/ethanol/water yielded 16.16 ± 1.04% and 15.68 ± 0.73%, respectively, values comparable to those obtained with the convention a chloroform/methanol/water (16.70 ± 0.07%) and significantly higher than the yield achieved with hexane (5.61 ± 0.33%). The extracted microbial oil, regardless of the solvent systems, showed a predominance of palmitic (C16:0), palmitoleic (C16:1), oleic (C18:1) and linoleic (C18:2) fatty acids. The iodine and saponification index, and acidity value were consistent with those found in commonly used edible oils. Furthermore, thermal analysis revealed that the solvent type influences the crystallization and melting behavior of the oils, possibly due to variations in the composition of triacyclglycerols (TAGs), free fatty acids (FFAs) and polar compounds. Therefore, our findings demonstrate that replacing highly toxic solvents with safer alternatives does not compromise the efficiency or quality of lipid extraction from <i>Y</i>. <i>lipolytica</i> NRRL Y-1095. Moreover, microbial oil exhibits potential for food and biotechnological applications due to its favorable composition and physicochemical properties.</p> Graphical abstract <p></p>